The Central Dogma and Junk DNA
On September 19, 1957, Francis Crick delivered a lecture during a symposium at University College London, titled “Protein Synthesis”. The lecture was published a year later (1); in it, Crick quotes his colleague James Watson as saying, “The most significant thing about the nucleic acids is that we don’t know what they can do.” In contrast, Crick argued that proteins play a central, indispensable role as enzymes within the cell that catalyze a variety of chemical reactions. He believed that the main role of genetic material was to control the synthesis of proteins, although the mechanism of that process was not known.
Crick’s hypothesis came to be known as the central dogma of molecular biology, and it was immortalized in his hand-written notes that described the flow of information from DNA to RNA to proteins. This achievement was all the more remarkable, considering that messenger RNAs were completely unknown at that time, and very little was known about how the cellular translational machinery functioned within the cytoplasm to synthesize proteins. Although the later discovery of retroviruses appeared to challenge Crick’s central dogma, he explained quite succinctly that his original statement had simply been misunderstood, and that information could flow in both directions between DNA and RNA (2).
Crick’s concept of information transfer led to a comparison of the genome to computer code—a set of instructions for the assembly of proteins, seen as the building blocks of the cell. Genome sequencing efforts, culminating in the completion of the human genome project, revealed that less than 2% of the human genome consisted of protein-coding genes. As early as the 1960s, scientists had referred to the vast majority of noncoding genomic sequences as “junk DNA” (3). Many of these sequences have accumulated—over several million years of evolution—from DNA recombination, from other species’ genomes, and even from ancient viruses.
Rapid advancements in sequencing and gene expression analysis technology led to another global, multidisciplinary project to understand the functional elements of the human genome: the ENCODE Project. The initial publication of the consortium’s results included a statement that 80% of the noncoding genomic regions were assigned a biochemical function (4). These results provoked a debate about the true nature of “junk DNA” that continues today.
“Junk is not garbage,” wrote Palazzo and Koonin in a Cell perspective (5). Their argument focused on a class of RNAs known as long noncoding RNAs (lncRNAs). LncRNAs are, somewhat arbitrarily, defined as RNAs longer than 200 nucleotides (nt) that are not translated into proteins. The 200nt cutoff serves as a distinction from other types of smaller noncoding RNAs, such as microRNA (miRNA), small interfering RNA (siRNA) and small nucleolar RNA (snoRNA). Currently, almost 18,000 human lncRNA genes have been identified. We now know that lncRNAs are involved in a variety of biological functions. Recent research has highlighted a few of these functions and also offers a novel, lncRNA-based approach to therapeutic development.
LncRNA and Oncogenesis
Bi et al. investigated the role of lncRNA plasmacytoma variant translocation 1 (lnc-PVT1) in human gliomas (6), which constitute over 80% of all malignant brain tumors (7). Although lnc-PVT1 transcription is linked to a variety of cancers (6), the precise networks of miRNAs and mRNAs with which lnc-PVT1 interacts is not clear. Bi et al. wanted to define the precise role of lnc-PVT1 in glioma development. Based on prior research showing the involvement of the miRNA, miR-1207-3p, they focused on the mechanism of this lncRNA-miRNA interaction in the regulating expression of the hepatocyte nuclear factor-1 beta gene (HNF-1B) in gliomas. From microarray and qPCR analysis of glioma tissues and cell lines, they determined that lncPVT-1 transcription was upregulated and played an important role in cell proliferation, migration, invasion and angiogenesis. Using bioinformatics analysis, the researchers predicted the regulatory networks affected by the lncPVT-1/miR-1207-3p/HNF-1B axis. These predictions were confirmed using the Dual-Luciferase® Reporter Assay System in cotransfection experiments with wild-type and mutant lncRNA-PVT1 and HNF1B expression vectors. The results suggest that the lncPVT-1/ miR-1207-3p/HNF-1B axis may present an attractive target for glioma therapeutic development.
LncRNA and the Inflammasome
Inflammasomes are intracellular protein complexes that are involved in activation of proinflammatory cytokines, such as interleukin 1β. The NLRP3 inflammasome has been widely studied, and its dysregulation is implicated in a variety of diseases. Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are associated with endothelial barrier injury, and these diseases can further trigger the activation of inflammatory cytokines through NLRP3 inflammasome activation (8).
Sun et al. investigated the role of an miRNA and a lncRNA in the pathology of ALI/ARDS (9). miR-223 was previously shown to inhibit NLRP3 inflammasome activation in cell culture. The lncRNA opa-interacting protein 5 antisense RNA 1 (OIP5-AS1) promotes inflammation and apoptosis in cells and also causes endothelial cell injury (9). The present study showed that lncRNA OIP5-AS1 and miR-223 were both dysregulated in the serum of patients with ARDS/ALI, compared to healthy donors. Using an in vitro model of ALI/ARDS in human pulmonary microvascular endothelial cells (HPMECs) treated with lipopolysaccharide (LPS), the researchers showed that miR-223 levels were decreased, while OIP5-AS1 levels were increased, in LPS-treated HPMECs. After bioinformatics analysis to predict interactions between miR-223 and OIP5-AS1, experiments using the Dual-Luciferase® Reporter Assay System confirmed that knockdown of OIP5-AS1 promoted the expression of miR-223. Conversely, overexpression of OIP5-AS1 inhibited the expression of miR-223, suggesting that miR-223 is a direct target of OIP5-AS1. Further studies demonstrated that miR-223 overexpression also promoted proliferation and inhibited apoptosis, pyroptosis, inflammatory response and oxidative stress of LPS-treated HPMECs; these effects were abolished by NLRP3 overexpression. Therefore, OIP5-AS1 knockdown and miR-223 overexpression could guide therapeutic approaches to treating ALI/ARDS.
Therapeutic development using RNA-directed approaches has gained popularity due to the relative ease of synthesis and manufacturing scale-up, compared to traditional small-molecule or monoclonal antibody therapies. Current mRNA vaccines, now a household term, exemplify the advantages of this approach. Although antisense oligonucleotides are the most common RNA-based therapeutic approach, recent attention has turned to lncRNAs (10). However, the comparatively large size of lncRNAs poses a major obstacle when it comes to delivering them into a cell. By identifying the functional regions of a lncRNA molecule, it may be possible to synthesize a smaller “lncRNA mimic” to overcome this challenge.
Li et al. used this strategy to study the inherited metabolic disorder phenylketonuria (PKU) in a mouse model (11). PKU is caused by multiple variants of the phenylalanine hydroxylase (PAH) gene that result in PAH deficiency. The researchers found that a mouse lncRNA Pair and human lncRNA HULC both associated with PAH. Pair knockout mice exhibited high levels of blood phenylalanine and symptoms that modeled human PKU. Further, HULC depletion led to reduced PAH enzymatic activities in human differentiated induced pluripotent stem cells. LncRNA mimics of Pair and HULC were successful in reducing excess phenylalanine levels in liver and improving phenylalanine tolerance in this mouse model.
The results described by Li et al. also overcame another drawback of lncRNA therapy: the poor sequence conservation between mouse and human lncRNAs, which makes it difficult to develop therapeutics in animal models that will be effective in the clinic. LncRNA mimics designed to match conserved lncRNA functional motifs, as in this study, could prove a viable alternative to other RNA-based therapeutic strategies.
The function of many lncRNAs remains unknown. Future research will undoubtedly reveal more roles for these molecules, adding credence to the belief that junk is, indeed, not garbage.
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- Crick, F.H.C. (1958) On protein synthesis. Symp. Soc. Exp. Biol. 12, 138–163.
- Crick, F. (1970) Central dogma of molecular biology. Nature 227, 561–563.
- Palazzo, A.F. and Gregory, T.R. (2014) The case for junk DNA. PLoS Genet. 10(5), e1004351.
- The ENCODE Project Consortium. (2012) An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74.
- Palazzo, A.F. and Koonin, E.V. (2020) Functional long non-coding RNAs evolve from junk transcripts. Cell 183, 1151–1161.
- Bi, Y. et al. (2021) LncRNA-PVT1 indicates a poor prognosis and promotes angiogenesis via activating the HNF1B/EMT axis in glioma. J. Cancer 12(19), 5732–5744.
- Goodenberger, M.L. and Jenkins, R.B. (2012) Genetics of adult glioma. Cancer Genet. 205, 613–621.
- Bos, L.D. et al. (2018) ARDS: challenges in patient care and frontiers in research. Eur. Respir. Rev. 27(147), 170107.
- Ji, J. et al. (2021) LncRNA OIP5-AS1 knockdown or miR-223 overexpression can alleviate LPS-induced ALI/ARDS by interfering with miR-223/NLRP3-mediated pyroptosis. J. Gene Med. (Preprint ahead of publication) https://doi.org/10.1002/jgm.3385
- Perry, R. B.-T. and Ulitsky, I. (2021) Therapy based on functional RNA elements. Science 373(6555), 623–624.
- Li, Y. et al. (2021) A noncoding RNA modulator potentiates phenylalanine metabolism in mice. Science 373(6555), 662–673.