Husna, Amity University Kolkata
RNA determines the identity of the cell and directs the responses to the cellular needs of the body. These diverse cellular functions are possible due to the vast chemical composition of RNA which comprises four ribonucleotides (A, C, G, and U) and more than 140 modified ribonucleotides. Several years of RNA research have laid the foundation for the development of RNA therapeutics including the varying antisense oligonucleotide therapy for spinal muscular atrophy, and mRNA vaccines. These striking achievements were possible because of the modified ribonucleotides, however, the ‘true’ RNA sequence, i.e., the ‘RNome’, remains unknown.
The term RNA sequencing is misleading; instead, a more accurate term is complementary DNA (cDNA) sequencing, because RNAs are converted back to DNA by reverse transcription and then they are sequenced. However, in the conversion of RNA-to-DNA, some crucial information on nucleotide modifications is lost. RNA sequences along with their modifications are the ‘true’ information content of RNA, therefore, RNome is required to usher in an era of molecular and clinical studies.
Why are true RNA sequences needed?
• None of the existing technology can determine the identity and position of all modifications at once in the full-length RNAs of both the single-molecule and at the transcriptome-wide scales.
• The reliability of cDNA sequences has led to a failure in obtaining and understanding the main regulatory codes within the RNome of human cells and other organisms, including many infectious viruses with RNA genomes.
• Direct sequencing of RNA would reveal the dynamics of base and sugar modifications as well as provide information on the locations of the modifications and splice sites, which will help in the basic understanding of how nucleic acids regulate cellular and organismal function and how dysregulation leads to diseases.
• The defects in RNA modifications account for more than 100 human diseases, including childhood-onset multiorgan failures, cancers, and neurologic disorders. These disorders are now known as ‘RNA modopathies’.
• As RNA viruses replicate and mutate quickly, obtaining the ‘true’ RNA sequences would be valuable in the early phases of disease outbreaks. Direct sequencing of RNA would also help the molecular epidemiologists to determine mutation types, propagation patterns, and an understanding of how the new viruses arise which will help to design effective eradication programs.
The recent study: A recent study by Nature Genetics has called for technological advancement. New technology is needed because there are some limitations in the two direct methods (mass spectrometry and nanopore technology), used for identifying the positions and types of modification in RNA. The liquid chromatography and tandem mass spectrometry (LC-MS/MS) method is considered the ‘gold standard for analysis of RNA modifications because it directly measures RNA. However, it can’t identify weakly abundant transcripts within a population, and it can’t even sequence longer RNA fragments. A larger challenge to this method is the lack of robust and specific nucleases which makes it difficult for fragment generation. Moreover, processing of the result of LC-MS/MS data requires additional informatics.
Nanopore sequencing is a promising technology that can analyze nucleic acids as single molecules (approximately 20 kb). This technology involves the detection of ion currents that are generated, in the context of a polynucleotide chain, as single nucleotides pass through small pores. Its base-calling is achieved by neural networks, however, direct base-calling can generally yield errors as high as 10–15%, so it poses a greater challenge for RNA sequencing. Moreover, the technology reads signals from several nucleotides at a time and therefore it has not achieved single-nucleotide resolution.
Significance of the study: It has called for an investment of funds and infrastructure to develop technologies for sequencing full-length RNA. Instruments that can sequence RNA directly and computational methods which can support those instruments as well as help in the analysis of the results are all needed. Undoubtedly, these resources, technology, and informatics necessary for direct RNA sequencing would be on the scale of the Human Genome Project. It would be an achievement in understanding advanced gene regulation and also lead to new frontiers in the field of health and medicine. Hence, mutual efforts are needed for the development of the technologies that are crucial for sequencing RNAs in a way that can preserve and even read the modifications.
Also read: The inevitable Coronavirus third wave in India!
Reference: Alfonzo, J.D., Brown, J.A., Byers, P.H. et al. A call for direct sequencing of full-length RNAs to identify all modifications. Nat Genet (2021). https://doi.org/10.1038/s41588-021-00903-1
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About the Author: Husna is an undergraduate student of BTech Biotechnology at Amity University Kolkata. She is a research enthusiast in Immunology and Immunotherapy but she has a keen interest in various other Bioscience subjects as well. She is constantly focused on improving her knowledge and laboratory skills through various internships. She is a Scientific content writer who knows diverse backgrounds in Biotechnology.
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