Currently, we have identified more than 170 RNA modifications, and these post-transcriptional modifications are widely present in various RNA molecules and are crucial for RNA function. In order to explore more dynamic changes of RNA modifications and their impact on cellular physiological functions, RNA modification sequencing technology has emerged. RNA modification sequencing technology can identify RNA modifications occurring on RNA molecules and map the modifications.Some conventional NAR modification sequencing techniques such as two-layer cellulose thin-layer chromatography, mass spectrometry, and high-performance liquid chromatography can identify and quantify RNA modifications, but they cannot precisely localize RNA modifications. In recent years, the latest development of high-throughput sequencing technology can carry out the precise location of RNA modification and low abundance of RNA molecules modification detection, and even can be tens or hundreds of thousands of RNA molecules at the same time at the same time, greatly improving our work efficiency, it has been widely used in a variety of biological tissues or cells in the detection of RNA modification. This paper mainly introduces the principle of high-throughput RNA modification sequencing technology and its application.
Chemical-assisted sequencingtechnologies
- Chemical processing is mainly applicable to the detection of RNA modifications in low abundance RNA species, and can be used to distinguish between modified and unmodified nucleotides in three main ways: installing biotin tags to enrich modified transcripts; inducing base mispairing or truncating transcripts during reverse transcription by altering base characteristics; and chemically inducing cleavage followed by linkage through specific articulators. The main chemical processing methods include Borohydride Reduction Sequencing (BoRed-seq),and Inosine Chemical Erasure Sequencing (ICE-seq).
- BoRed-seq is an innovative method to analyze RNA modifications by reducing RNA methylation modifications through borohydride and combining it with high-throughput sequencing technology. The core principle of BoRed-seq is to use borohydride to reduce and remove methylation modifications (e.g., m6A) from RNA molecules, and then analyze the processed RNAs through high-throughput sequencing. By comparing the differences between samples before and after treatment, BoRed-seq is able to precisely locate and quantify different methylation modification sites, thus revealing the role of RNA modifications in the regulation of gene expression and cellular functions. This method is highly specific and sensitive, enabling genome-wide RNA modification analysis. For example, extracted total RNA is treated with NaBH4, after which the treated RNA is exposed to low pH to produce debasement sites at the modification sites, such as m7G, which can then be labeled with biotin molecules to further reduce the concentration or activity of streptavidin.
- Inosine chemical erasure sequencing (ICE-seq) is a highly efficient technique for studying inosine (Inosine, I) modifications in RNA.ICE-seq chemically specifically erases inosine modifications from RNA, and subsequently analyzes the differences in RNA sequences before and after the erasure using high-throughput sequencing technology. The core of the method is to remove inosine from RNA molecules using chemical reagents such as acrylonitrile, and then prepare RNA-seq cDNA libraries of untreated and treated samples, isolate 400-500 bp fragments by agarose gel electrophoresis, and PCR amplify them for in-depth sequencing, as the G-base derived from I-base on the chemically-treated RNAs is efficiently eliminated to form N1-cyanoethylpyrimidine (ce1I), which is a blocker for N1-cyanoethylpyrimidine. ( ce1I ) blocked reverse transcription and its cDNA was truncated, so that the unerased inosine site could be recognized and enriched. By comparing data before and after the modification, ICE-seq is able to precisely localize inosine-modified sites and investigate their function in gene expression, RNA splicing and translation. The technique can also be applied to other RNA modifications including pseudouridine (Ψ) and 1-methyladenosine ( m1A ), m5C, and others.
- m6A-label-seq is a high-throughput sequencing technique for studying RNA methylation modifications (specifically N6-methyladenosine, m6A). By introducing specific labels (usually molecules containing radioactive or fluorescent tags) into RNA molecules, combined with high-throughput sequencing, the method is able to accurately identify m6A modification sites in RNA, thus enabling quantitative and qualitative analysis of m6A modifications on a genome-wide scale. For example, intracellular S-adenosylmethionine (SAM), a cofactor of methyltransferases, can form m6A by adding a methyl group to the N6 position of adenine under the action of m6A methyltransferases (METTL3/METTL14). SAM is synthesized from methionine and adenosine triphosphate (ATP), catalyzed by methionine adenyltransferase (MAT). Starting from the methylation source, methionine with allyl modification is introduced to generate the allyl-substituted cofactor allyl-SAM or the seleno homolog allyl-SeAM in the presence of intracellular MAT. intracellular METTL3/METTL14 can utilize either allyl-SAM or allyl-SeAM by allyl-adding its allyl group to the mRNA that originally is m6A site to obtain a6A (N6-allyl adenine). a6A is induced under mild iodine addition conditions to generate cyclized 1,6-position cyclized adenine (cyc-A), and then RNA cyc-A undergoes base mismatches during reverse transcription into cDNA. Finally, analyzing the mutation site with the help of high-throughput sequencing and bioinformatics, a single-base resolution map of the m6A site of the whole transcriptome can be obtained.
Antibody-based sequencing technologies
- Antibody-based sequencing technology can detect a variety of RNA modifications including m6A, m1A, m7G, hm5C and ac4C, but the basic principle of each sequencing technology is consistent, which is based on the immunoprecipitation of a specific RNA modification with its specific antibody combined with high-throughput sequencing RNA sequencing technology.In this technique, the total RNA of the tissue or cell to be detected is first extracted. After the RNA is purified, the specific antibody modified by the RNA to be detected is added for immunoprecipitation to enrich the RNA modified by the specific RNA. The enriched RNA is fragmented into 100-200bp, and then inverted to cDNA to construct libraries for high-throughput sequencing and combined with bioinformatics analysis tools to analyze and identify RNA modification.
Schematic diagrams and sequencing features of different technologies(Zhang Y et al., 2022)
Enzyme/Protein-Assisted Sequencing
Some enzymes or proteins and proteins associated with RNA modifications can also be used to capture or edit specific RNA modifications, allowing us to analyze specific transcripts to obtain RNA modification transcriptional profiles or to characterize the dynamics of RNA modifications.
- m6A-SEAL-Seq was used to study N6-methyladenosine (m6A) modification in RNA. fTO enzyme, as a demethylase, can oxidize the m6A modification in RNA to the highly reactive intermediate state product, N6-hydroxymethyladenine (hm6A), and then utilize the sulfhydryl group of dithiothreitol (DTT) to undergo imine addition reaction with hm6A, which converts the unstable The free sulfhydryl group present on dm6A can react with methanethiosulfonate (MTSEA) to couple the m6A modification site on the RNA with the labeled biotin, and the final modified transcript can be captured by streptavidin beads, thus enriching the RNA fragments containing the m6A modification. The final modified transcript can be captured by streptavidin beads, thus enriching RNA fragments containing m6A modifications for subsequent high-throughput sequencing. m6A-SEAL-Seq can efficiently and specifically identify m6A modifications with high sensitivity, enabling precise quantification and localization of m6A modifications throughout the genome, and has been widely used in the study of RNA methylation, regulation of gene expression, and the exploration of disease mechanisms. For example, researchers utilized m6A-SEAL-Seq to detect m6A modifications on polyA+ RNA from human HEK293T cells, and identified 8605 high-confidence m6A sites, which conformed to the conserved sequence (RRACH) and distribution pattern of m6A.
- MAZTER-seq (m6A-sensitive RNA endonuclease-facilitated sequencing) relies on the recognition of the ACA motif on RNA by the RNA endonuclease MazF, a method capable of detecting m6A-modified sites and their abundance with single-base resolution across the entire transcriptome.MazF is an RNA endonuclease capable of specifically cleaving the 5' end of the ACA motif on RNA without enzymatically cleaving the (m6A)CA motif with m6A modification.Therefore, this property of MazF of being able to cleave a certain segment of RNA with an ACA sequence can be utilized to identify whether or not that segment of RNA has an m6A modification.Because the m6A modifying motif on RNA is generally RRACH (R=A/G, H=A/C/U), and RNA sequences treated with MazFase contain an ACA motif at the 5' end, the (m6A)CA motif within the RNA can be recognized as a methylation site. However, this method is limited by the specific selectivity of endonuclease for RNA sequences, which prevents RNA sequences that do not contain the m6A CA motif from being recognized.
- DART-seq (neighboring RNA modification deamidation targeting by sequencing) is an antibody-independent method for detecting m6A modification sites in transcripts by means of an exogenous APOBEC1-YTH fusion protein. The cytidine deaminase APOBEC1 can bind to the m⁶A-binding structural domain YTH, which recognizes m6A modification sites, and the expression of APOBEC1 attached to YTH induces the editing of C to U, i.e., C-to-U deamination, on cytosine residues in the vicinity of m⁶A modification sites. These editing sites were identified as m6A-modified regions in subsequent data analysis. Since the binding of the YTH structural domain to the 2'-OH of the m6A-modified residues via hydrogen bonding is the key structure, the YTH structural domain in DART-seq could not recognize m6Am, ensuring specificity. Although DART-seq is capable of identifying m6A sites and mapping m6A modifications from low amounts of RNA (~10 ng of total RNA), the need to transiently transfect APOBEC1-YTH fusion proteins into mammalian cells, which potentially disrupts the physiological state of the cells, has also limited its application.
RNA modifications nanopore sequencing
- RNA modified nanopore sequencing technology is a technology based on nanopore sequencing platform, which can directly analyze and identify chemical modifications ( such as methylation, pseudouridine, etc. ) in RNA molecules. This technology combines the high resolution of nanopore sequencing and the sensitivity to RNA modification, and has the advantages of direct sequencing, real-time detection and long read length ( up to 2Mb ), and does not require additional chemical treatment or labeling. The basic principle is as follows : Nanopore sequencing decodes RNA molecules passing through pores ( usually nanoscale pores ) through current changes. Whenever the RNA molecule is pulled by the motor protein through the nanopore, it will cause a small change in the current. The magnitude and pattern of the change can reflect the sequence of the molecule. The charged properties of each nucleotide ( A, U, C, G or other RNA bases ) are different. Different bases cause different current change patterns when passing through the nanopore. These changes can be measured and used to infer the sequence of nucleotides.Different modifications on RNA will lead to changes in the structure and charge characteristics of the modified bases and the original bases. When a single modified RNA molecule passes through the nanopore, RNA modification changes the current characteristics of the molecule. Therefore, different types of RNA modification will leave specific ' fingerprints ' in the current map. By analyzing these current signals, we are able to identify different types of modifications in RNA molecules. Each RNA modification causes different degrees of current changes, and the type and location of the modification are closely related to the mode of the current signal. By comparing with the database of known modification types, the type and location of modification can be determined. Nanopore sequencing has a high time resolution and can detect RNA molecules passing through the pores in real time. For some rapidly changing RNA modifications or RNA modifications that are difficult to detect by traditional methods, nanopore sequencing has great advantages.
For a more in-depth understanding of RNA Modifications, refer to "Overview of RNA Modifications."
Schemes of nanopore direct sequencing-based detection technology(Zhang Y et al., 2022)
References:
- Zhang Y, Lu L, Li X."Detection technologies for RNA modifications."Exp Mol Med.2022;54(10):1601-1616.https://pubmed.ncbi.nlm.nih.gov/36266445/
- Wang Y, Xiao Y, Dong S, Yu Q, Jia G."Antibody-free enzyme-assisted chemical approach for detection of N6-methyladenosine."Nat Chem Biol.2020;16(8):896-903.https://pubmed.ncbi.nlm.nih.gov/32341502/
- Okada S, Ueda H, Noda Y, Suzuki T."Transcriptome-wide identification of A-to-I RNA editing sites using ICE-seq."Methods. 2019;156:66-78.https://pubmed.ncbi.nlm.nih.gov/30578846/
- Shu X, Cao J, Cheng M, Xiang S, Gao M, Li T, Ying X, Wang F, Yue Y, Lu Z, Dai Q, Cui X, Ma L, Wang Y, He C, Feng X, Liu J."A metabolic labeling method detects m6A transcriptome-wide at single base resolution."Nat Chem Biol.2020;16(8):887-895.https://pubmed.ncbi.nlm.nih.gov/32341503/
