ncRNAs, once considered “junk DNA,” are now recognized as critical regulators of gene expression. Its clinical implementation auspices a new era of medicine
Background
The Human Genome Project (HGP) found in 2001 – the publication year of the human genome, to humanity’s surprise, around 75% of the genome is actively transcribed yet only 1.5% of the genome codes for proteins; roughly 98% of the genome produces non-coding RNAs. Prior to the initiation of HGP, the scientific community has already been acquainted with regulatory, non-coding regions in the genome; in particular, the alternative splicing mechanism in eukaryotes was discovered in 1977 by Richard J. Roberts and Phillip A. Sharp – they were subsequently awarded the Nobel Prize in 1993 for this discovery. This study highlights, with timeless vigor, the significance and complexity of non-coding regions in the genome. The diverse family of noncoding RNAs certainly exemplifies this fact.
Noncoding RNA (ncRNA) is a functionally conserved RNA molecule that is not translated into a protein but one that regulates gene expression at transcriptional and post-transcriptional levels. ncRNAs are typically classified by their length: small ncRNAs (<200 nucleotides) and long ncRNAs (>200 nucleotides). They are involved in regulation at both the epigenetic and genetic level. Their dysregulation correlates with disease. They are mysterious. They are everywhere.
Short ncRNAs
In 2006, the Nobel Prize in Physiology or Medicine was awarded to Andrew Fire, of Stanford University, and Craig Mello, University of Massachusetts Medical School, for their discover of RNA interference (RNAi). RNAi depicts the mechanism by which gene expression is inhibited through the action of short interfering RNA molecules (siRNAs) or microRNAs (miRNAs) – both are small ncRNAs).
Discovered in Caenorhabditis elegans – otherwise known as roundworms –, this mechanism can be both endogenously and exogenously induced, by miRNA and siRNA respectively. In the endogenous pathway, Drosha (an endonuclease) processes primary miRNA to a ~60-70 nucleotide long, looped, double stranded pre-miRNA. Exported into cytoplasm, the pre-miRNA is loaded onto an RNA-Inducing-Silencing-Complex (RISC). RISC is composed of two subunits: dicer and argonaut. The dicer trims the pre-miRNA into ~20-22 nucleotide long, double stranded fragments; a helicase then unwinds the RNA duplex, degrading one strand (the passenger strand) and allowing the other (the guide strand) to remain incorporated in the RISC. The guide strand (now the mature miRNA) will partially hybridize with an mRNA strand, leading the RISC to repress its translation. For siRNAs, the final step requires perfect rather than partial hybridization.
Long ncRNAs
Long non-coding RNAs (lncRNAs) can also conduct a variety of tasks. Notably, cis regulation – regulation of nearby genes, is done by lncRNAs interacting with nearly activators and repressors; impressively, they can permeate beyond the local site, to the whole chromosome.
Additionally, lncRNAs conduct trans-chromatin interaction, where they can influence gene expression across chromosomes. The critical inactivation of the X chromosome relies heavily on lncRNA activity – specifically Xist: the chromatin structure is altered in a stepwise fashion, mediated by Xist commuting with repressors and chromatin structural regulators, repressing most genes on the X-chromosome.
In 2012, the ENCODE project initiated the characterization of ncRNAs. In more recent years, the NONCODE database and FANTOM consortium, though yielding different results, both aim to profile and document lncRNAs in human cells, propelled by developments in machine learning models and next-generation sequencing techniques. Nevertheless, our understanding of lncRNAs remain excruciatingly opaque.
Clinical Applications
ncRNAs are primarily used as diagnostic biomarkers for diseases. miRNAs and lncRNAs can be detected in blood, urine, or tissue biopsies. Their dysregulated expression levels correlate with disease presence or progression. Certain ncRNAs, such as miR-124, is crucial for neuronal differentiation and synaptic function, is significantly downregulated in HD. As aforementioned, ncRNAs are apt gene silencers and enhancers, sometimes occupying the chokepoints for certain pathways implicated in pathology. Thus, ncRNA-based therapies intuitively target and repress harmful gene expressions or reverse harmful gene silencing. These modulating ncRNAs are also used in combinatory treatments with established treatments such as chemotherapy and biologics to enhance efficacy. Since the HGP, personalized medicine has been a hallmark of future clinical approach, and ncRNAs seem to possess its own crucial role in this field. Analyzing the patient’s transcriptome profile, focusing on certain signature regions, can help tailor therapies which address an individual’s needs more specifically and efficiently. This method has been combined in multi-omic approaches, with genomics and proteomics, to further refine the accuracy of treatments.
Conclusion
Evidently, as demonstrated by the HGP, the sophistication of the human genome lies far beyond its prolific repertoire of proteins, but more so in its voluminous collection of non-coding regions, which serve a plethora of purposes, including regulation of gene expression, structural organization, epigenetic regulation, and more. ncRNAs exemplify this versatility and “omnipotence” of non-coding regions. Currently, various clinical approaches envision harnessing the potential of ncRNAs, particularly as ncRNAs possess the potential to become a malleable tool for clinical use – like the CRISPR/Cas system. However, issues such as off-target effects, delivery, and efficiency remain as barriers to its clinical success. Continued interdisciplinary efforts will be essential to understanding ncRNAs as well as overcoming these obstacles. The non-coding regions of our genome, once dismissed as 'junk,' is shedding light unto previously overcast areas in health. Engineering and utilizing such technology is not only destined to revolutionize our understanding of medicine but more importantly, our comprehension of the very mechanisms of life.
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