Why is Non-coding RNA Hot in Biotechnology

Since 2010 over 250,000 research papers have been published on non-coding RNA (ncRNA) [1]. Of relevance to biotechnology, nearly 90,000 of those papers discuss ncRNA and disease, and 70,000 discuss using ncRNA as therapeutic agents (drugs) or being drug targets. The following sections introduce the drivers for ncRNA interest, companies working on ncRNA, skills needed to work in ncRNA companies, and ncRNA classification and function. 

Why is non-coding RNA getting so much attention?

Small molecule and biologic drugs predominately target proteins. Among the total amount of the human genome that is transcribed into RNA, proteins are translated from a very small fraction. Indeed, a surprise of the human genome project was that only about 20,000 genes, corresponding to approximately 45Mb (megabases) of DNA, or 1.5% of the 3Gb (gigabase) haploid human genome, encode protein sequences [2].  When the size of the “druggable” genome is estimated, the fraction of the genome targeted by drugs is even smaller.

The druggable genome is defined as the number of proteins that are known to cause disease and sites within those proteins where drugs can bind and cause an action such as inhibit an enzyme’s function, block a receptor / ligand interaction, or alter a protein’s shape (allosteric modification). Several estimates indicate that between 10% and 15% of known proteins are involved in disease and only 3.5% of this fraction currently is drugged [3]. In numbers, FDA approved drugs target between 700 and 900 proteins. In other words, only 0.05% of the human genome is currently targeted by existing drugs [4]. What about the other 99.95%?

Turns out, a large fraction - 70% - of the human genome is transcribed into RNA [5]. The vast majority of this RNA is non-coding. Because ncRNA regulates gene expression at both transcriptional and translational levels, it can be harnessed in targeted ways to act as a drug. Moreover, RNA molecules fold into structures that can bind small and large molecules. Thus, RNA can be both a drug and a drug target, representing a new frontier in biotechnology.

The therapeutic potential of ncRNA is significant. Pubmed searches of non-coding RNA and disease or therapeutics show that ncRNA has roles in immunity, cancer, neurobiology, diabetes, and many other diseases and syndromes including infection [6]. In addition to therapies, ncRNAs may be useful as diagnostic biomarkers, because the levels of specific ncRNAs change in response to disease or infection [7]. Beyond applications in human health, ncRNA is being explored in novel ways that include biosensors [8] and RNA-based enzymes (ribozymes) [9].

Who works on non-coding RNA and what skills are needed?

The Biotech-Careers.org employer database reflects biotechnology’s interest in RNA. As of Feb 6, 2023, 129 employers in 214 locations have “RNA” as one their business areas.* These employers work in 99 other areas. The top ten areas include Therapeutics, COVID-19, Vaccines, RNAi, Small Molecules, Cell and Gene Therapy, DNA, mRNA, Bioinformatics, and Diagnostics. In the case of diagnostics, many of the companies working on Liquid Biopsy are looking into biomarkers based on ncRNA packaged in exosomes (exRNA, below).

To work with these employers it is important to understand aspects of molecular biology that include transcription, translation, and base-pair complementarity.  Knowledge of ncRNA diversity and function is beneficial. Hands-on experience working with RNA, using in vitro transcription systems, and fluorescence-based assays is important.

What to non-coding RNA’s do?

As discussed, RNA either encodes proteins or is non-coding. Despite the name, non-coding RNA is highly functional and has roles in translating mRNA into protein and regulating gene expression and function at transcriptional, translational, and protein levels. Non-coding RNAs also forms the catalytic domains in ribosomes [10], self splicing introns [11], and other protein complexes. The activity of ncRNA is typically local to the cells where the molecules are produced (intracellular activity), but ncRNA molecules can also be packaged into lipid vesicles or protein complexes and sent off to exert their activity in other cells (exRNA, extracellular activity). These extracellular vesicles can even shed into the environment and regulate genes in other organisms [12]. 

Translation

In translation, ribosomes, the molecular machines that convert the mRNA code into protein sequences, utilize ribosomal RNA (rRNA) as a scaffold for assembling ribosomal proteins and linking amino acids together into a protein sequence [10]. Transfer RNA (tRNA) brings amino acids to the ribosome. Each tRNA carries a specific amino acid and “pairs” with the mRNA codons via its complementary anticodon loop. It is worth noting that tRNA’s are processed into mature forms by RNase P, a ribonucleoprotein (RNP) complex, which uses a ribozyme to cleave off a precursor sequence [13].

HC Bioscience is developing tRNA based drugs to "fix" proteins that are too short due to mutations that introduce stop codons, and to "mark" disease causing proteins for degradation. In the case of short proteins, their PTCX (“Patch”) platform uses engineered tRNAs to recognize nonsense mutations in mRNA transcripts and insert an amino to restore the protein to its full length and function in analogy to suppressor mutations. Their second platform, called SWTX (“Switch”) targets diseases caused by unwanted protein. SWTX uses tRNAs that map to rare codons to introduce amino acids that presumably create sites that are recognized by proteases.

In addition to protein synthesis, other translation related ncRNAs are involved in removing introns  between the exons (splicng) in pre-mRNA (heteronuclear (hnRNA)) to its mature form. These RNA’s, known as small nuclear RNAs (snRNA), form a complex with proteins (the spliceosome) and bind to specific sequence elements within introns [14].  Several snRNAs (called U1-U7 due to their high uracil content) participate in a multi step reaction involving different snRNA molecules. These molecules gain additional properties through modifications in which various bases are methylated or converted to pseudouridine.

In addition to snRNA’s, tRNA, mRNA, and other RNAs can be extensively modified. To date, over 150 modified RNA base modifications have been identified [15]. Pseudouridylation [16] is a common modification where the glycosidic bond (attaches uridine to the ribose sugar) of uridine (U) is isomerized from an N-C bond (C1 of the ribose to N1 of uridine) to a C-C bond (C1 of the ribose to C5 of uridine). Pseudouridylation is carried out by protein only complexes and small nucleolar RNA (snoRNA)-dependent RNPs. The later case is important in modifying snRNA’s prior to splicing.

Pseudouridine (ψ) enhances RNA stability and decreases anti-RNA immune responses. A significant part of the success of the Pfizer-BioNTech and Moderna vaccines is attributed to the fact that the U’s in the SARS-CoV-2 spike protein mRNA are replaced with N1-methyl-ψ. When compared the Curevac vaccine, for which the mRNA did not contain ψ, the  Pfizer-BioNTech and Moderna vaccines were over 90% effective, whereas the CureVac vaccine was only 48% effective [17]. All three vaccines were formulated in the same way the only differences being in wether U was replaced with ψ.

Regulation

Regulatory ncRNAs are numerous and diverse. Most ncRNAs are transcribed from the genome and many are transcribed from introns within protein encoding genes. Some, in the cases of tRNA derived small RNA (tsRNA) and circular RNA (circRNA), arise from RNA degradation and processing [18, 19]. Similarly, some lncRNAs are processed in RNAi pathways into mi and siRNAs [20].

Originally, ncRNAs were thought to be small (100 nucleotides (nts) or less). As DNA sequencing advanced, RNA could be studied at large scale and a new class of ncRNA, long non-coding (lncRNA), was defined by virtue of being longer than 200 nts [21]. So, ncRNAs range in size from 10’s of nts to several 1000 nts. Just as they have a wide range of sizes they also have a wide range of activities and in work in protein-dependent and protein-independent.

A common ncRNA activity is to block protein translation. In the central-dogma paradigm of DNA being transcribed into RNA, and RNA translated into protein, by convention, the RNA that is translated (mRNA) corresponds to the sense strand of the double stranded DNA. The complement of the sense strand is the antisense strand. It is important to note that this distinction is localized within protein encoding genes, and that DNA can, and is, transcribed from both strands. In the case of cis-antisense RNA’s (cis-asRNA) the ncRNA is transcribed from the same gene that encodes a protein. Trans-asRNA’s come from elsewhere in the genome. Cis-asRNAs have higher specificity and tend to be shorter than trans asRNAs [22], but a single trans-asRNA can affect multiple genes.

Another large group of antisense RNAs belong to RNA interference (RNAi) pathways. RNAi pathways involve multiple enzymes and proteins that prepare different kinds of RNA (miRNA, siRNA, piRNA, rasiRNA) for interacting with target molecules. These RNAs begin as larger double stranded molecules that are processed and assembled into an RNA-induced silencing complex (RISC) that can interact with mRNA or DNA [23].

Last, Y RNAs were discovered in 1981 [24] by researchers studying lupus erythematosus. They were named Y RNA to distinguish them from the snRNAs which are called U1-U7. That's Y not U instead of why not Y. Y RNAs have a role in DNA replication and RNA processing and quality control [25]. Like other ncRNAs, Y RNAs and their fragments are being investigated as biomarkers and therapeutic targets in cancer, cardiovascular disease, and auto immunity [26, 27, 28].

Summary

The full impact and potential for non-coding RNA is an active area of basic and applied research. As high-throughput data collection technologies, like DNA sequencing, have improved, we can "see" deeper to understand how RNA is a key molecule in biological systems. Insights from large-scale data gathering experiments are uncovering new applications of RNA in human health and synthetic biology. In these applications, RNA is being harnessed as drugs, drug targets, and nanotechnologies such as biosensors.

More detail for the above descriptions is provided in the accompanying table. The table provides RNA species names with expanded acronyms, the average lengths of each species, estimated numbers in the human genome, RNA origin, RNA polymerase that transcribes the RNA, its role / comments, and additional references. 

 

*The number of companies working on RNA is frequently updated and may not match the number indicated in the blog.

References:

With one exception, citations focus on recent open access review articles and websites.  

[1] PubMed search with the terms “non-coding RNA”, “non-coding RNA disease”,  “non-coding RNA therapeutics”;  (https://pubmed.ncbi.nlm.nih.gov/?term=non-coding+rna), https://pubmed.ncbi.nlm.nih.gov/?term=non-coding+rna+disease, https://pubmed.ncbi.nlm.nih.gov/?term=non-coding+rna+therapeutics)

[2] Ezkurdia, I., Juan, D., Rodriguez, J. M., Frankish, A., Diekhans, M., Harrow, J., Vazquez, J., Valencia, A., & Tress, M. L. (2014). Multiple evidence strands suggest that there may be as few as 19,000 human protein-coding genes. Human molecular genetics, 23(22), 5866–5878. https://doi.org/10.1093/hmg/ddu309

[3] Warner, K. D., Hajdin, C. E., & Weeks, K. M. (2018). Principles for targeting RNA with drug-like small molecules. Nature reviews. Drug discovery, 17(8), 547–558. https://doi.org/10.1038/nrd.2018.93 - And cited references.

[4] Santos, R., Ursu, O., Gaulton, A., Bento, A. P., Donadi, R. S., Bologa, C. G., Karlsson, A., Al-Lazikani, B., Hersey, A., Oprea, T. I., & Overington, J. P. (2017). A comprehensive map of molecular drug targets. Nature reviews. Drug discovery, 16(1), 19–34. https://doi.org/10.1038/nrd.2016.230 - The article analyzed 2015 data. Since then more drugs, likely targeting addition proteins, have been approved.  

[5] Djebali, S., Davis, C. A., Merkel, A., Dobin, A., Lassmann, T., Mortazavi, A., Tanzer, A., Lagarde, J., Lin, W., Schlesinger, F., Xue, C., Marinov, G. K., Khatun, J., Williams, B. A., Zaleski, C., Rozowsky, J., Röder, M., Kokocinski, F., Abdelhamid, R. F., Alioto, T., … Gingeras, T. R. (2012). Landscape of transcription in human cells. Nature, 489(7414), 101–108. https://doi.org/10.1038/nature11233

[6] Smith, E. S., Whitty, E., Yoo, B., Moore, A., Sempere, L. F., & Medarova, Z. (2022). Clinical Applications of Short Non-Coding RNA-Based Therapies in the Era of Precision Medicine. Cancers, 14(6), 1588. https://doi.org/10.3390/cancers14061588

[7] Cheong, J. K., Rajgor, D., Lv, Y., Chung, K. Y., Tang, Y. C., & Cheng, H. (2022). Noncoding RNome as Enabling Biomarkers for Precision Health. International journal of molecular sciences, 23(18), 10390. https://doi.org/10.3390/ijms231810390

[8] Weaver, S., Mohammadi, M. H., & Nakatsuka, N. (2023). Aptamer-functionalized capacitive biosensors. Biosensors & bioelectronics, 224, 115014. https://doi.org/10.1016/j.bios.2022.115014

[9] Debiais M, Lelievre A, Smietana M, Müller S. Splitting aptamers and nucleic acid enzymes for the development of advanced biosensors. Nucleic Acids Res. 2020 Apr 17;48(7):3400-3422. doi: 10.1093/nar/gkaa132. PMID: 32112111; PMCID: PMC7144939.

[10] Simonović, M., & Steitz, T. A. (2009). A structural view on the mechanism of the ribosome-catalyzed peptide bond formation. Biochimica et biophysica acta, 1789(9-10), 612–623. https://doi.org/10.1016/j.bbagrm.2009.06.006

[11] https://en.wikipedia.org/wiki/Ribozyme

[12] Veziroglu, E. M., & Mias, G. I. (2020). Characterizing Extracellular Vesicles and Their Diverse RNA Contents. Frontiers in genetics, 11, 700. https://doi.org/10.3389/fgene.2020.00700

[13] Phan, H. D., Lai, L. B., Zahurancik, W. J., & Gopalan, V. (2021). The many faces of RNA-based RNase P, an RNA-world relic. Trends in biochemical sciences, 46(12), 976–991. https://doi.org/10.1016/j.tibs.2021.07.005

[14] Morais P, Adachi H, Yu YT. Spliceosomal snRNA Epitranscriptomics. Front Genet. 2021 Mar 2;12:652129. doi: 10.3389/fgene.2021.652129. PMID: 33737950; PMCID: PMC7960923.

[15] Boccaletto P, Stefaniak F, Ray A, Cappannini A, Mukherjee S, Purta E, Kurkowska M, Shirvanizadeh N, Destefanis E, Groza P, Avşar G, Romitelli A, Pir P, Dassi E, Conticello SG, Aguilo F, Bujnicki JM. MODOMICS: a database of RNA modification pathways. 2021 update. Nucleic Acids Res. 2022 Jan 7;50(D1):D231-D235. doi: 10.1093/nar/gkab1083. PMID: 34893873; PMCID: PMC8728126.

[16] Borchardt EK, Martinez NM, Gilbert WV. Regulation and Function of RNA Pseudouridylation in Human Cells. Annu Rev Genet. 2020 Nov 23;54:309-336. doi: 10.1146/annurev-genet-112618-043830. Epub 2020 Sep 1. PMID: 32870730; PMCID: PMC8007080.

[17] COVID: Morais P, Adachi H, Yu YT. The Critical Contribution of Pseudouridine to mRNA COVID-19 Vaccines. Front Cell Dev Biol. 2021 Nov 4;9:789427. doi: 10.3389/fcell.2021.789427. PMID: 34805188; PMCID: PMC8600071.

[18] George, S., Rafi, M., Aldarmaki, M., ElSiddig, M., Al Nuaimi, M., & Amiri, K. M. A. (2022). tRNA derived small RNAs-Small players with big roles. Frontiers in genetics, 13, 997780. https://doi.org/10.3389/fgene.2022.997780

[19] Liang, Y., Liu, N., Yang, L., Tang, J., Wang, Y., & Mei, M. (2021). A Brief Review of circRNA Biogenesis, Detection, and Function. Current genomics, 22(7), 485–495. https://doi.org/10.2174/1389202922666210331130722

[20] Atkinson SR, Marguerat S, Bitton DA, Rodríguez-López M, Rallis C, Lemay JF, Cotobal C, Malecki M, Smialowski P, Mata J, Korber P, Bachand F, Bähler J. Long noncoding RNA repertoire and targeting by nuclear exosome, cytoplasmic exonuclease, and RNAi in fission yeast. RNA. 2018 Sep;24(9):1195-1213. doi: 10.1261/rna.065524.118. Epub 2018 Jun 18. PMID: 29914874; PMCID: PMC6097657.

[21] St Laurent G, Wahlestedt C, Kapranov P. The Landscape of long noncoding RNA classification. Trends Genet. 2015 May;31(5):239-51. doi: 10.1016/j.tig.2015.03.007. Epub 2015 Apr 10. PMID: 25869999; PMCID: PMC4417002.

[22] https://en.wikipedia.org/wiki/Antisense_RNA

[23] Pratt AJ, MacRae IJ. The RNA-induced silencing complex: a versatile gene-silencing machine. J Biol Chem. 2009 Jul 3;284(27):17897-901. doi: 10.1074/jbc.R900012200. Epub 2009 Apr 1. PMID: 19342379; PMCID: PMC2709356.

[24] Lerner, M. R., Boyle, J. A., Hardin, J. A., & Steitz, J. A. (1981). Two novel classes of small ribonucleoproteins detected by antibodies associated with lupus erythematosus. Science (New York, N.Y.), 211(4480), 400–402. https://doi.org/10.1126/science.6164096 - Paywalled article.

[25] Kowalski MP, Krude T. Functional roles of non-coding Y RNAs. Int J Biochem Cell Biol. 2015 Sep;66:20-9. doi: 10.1016/j.biocel.2015.07.003. Epub 2015 Jul 6. PMID: 26159929; PMCID: PMC4726728.

[26] Gulìa, C., Signore, F., Gaffi, M., Gigli, S., Votino, R., Nucciotti, R., Bertacca, L., Zaami, S., Baffa, A., Santini, E., Porrello, A., & Piergentili, R. (2020). Y RNA: An Overview of Their Role as Potential Biomarkers and Molecular Targets in Human Cancers. Cancers, 12(5), 1238. https://doi.org/10.3390/cancers12051238

[27] Valkov, N., & Das, S. (2020). Y RNAs: Biogenesis, Function and Implications for the Cardiovascular System. Advances in experimental medicine and biology, 1229, 327–342. https://doi.org/10.1007/978-981-15-1671-9_20

[28] Boccitto, M., & Wolin, S. L. (2019). Ro60 and Y RNAs: structure, functions, and roles in autoimmunity. Critical reviews in biochemistry and molecular biology, 54(2), 133–152. https://doi.org/10.1080/10409238.2019.1608902