Sample Submission GuidelinesTransfer RNA (tRNA) 2'-O-Methylation: A Key Player in Biological Functions and Disease Mechanisms
Transfer RNA (tRNA) harbors an extensive array of post-transcriptional modifications crucial for exerting its biological functions. Among these, methylation stands out as the most prevalent type of tRNA modification, occurring either on the nucleotide bases or at the 2'-O-ribose position (2'-O-methylation). The 2'-O-methylation modification is widespread in archaea, prokaryotes, and eukaryotes, identified at positions 4, 6, 18, 32, 34, 39, 44, 54, and 56 within tRNA.
Research indicates that 2'-O-methylation plays a regulatory role in tRNA folding, maturation, stability, and the precision of anticodon-mRNA codon pairing. It is closely associated with cellular processes such as growth, stress response, and immune regulation. Deficiencies in cytoplasmic tRNA 2'-O-methylation are often linked to various diseases. Enzymes involved in tRNA 2'-O-methylation present potential targets for drug development.
Investigating tRNA 2'-O-methylation and its modifying enzymes will enhance our understanding of the biological functions of this modification. Furthermore, it lays the groundwork for exploring the pathogenic mechanisms of tRNA 2'-O-methylation enzymes in human diseases.
(Yuri Motorin, Virginie Marchand. Genes 2018)
2'-O-RNA Methylation Detection Service
CD Genomics offers a comprehensive 2'-O-RNA methylation detection service, designed to identify the presence of 2'-O-methylation modifications on RNA molecules and determine the specific sites of 2'-O-methylation. Beyond the offered service, CD Genomics has innovated a suite of products designed for the assessment of 2'-O-RNA methylation levels in diverse RNA molecules, encompassing mRNA, LncRNA, pri-miRNA, tRNA, and rRNA.
Deep Sequencing-Based Approaches for Detection of 2'-O-methylation
(Yuri Motorin, Virginie Marchand. Genes 2018)
Key Features of CD Genomics 2'-O-RNA Methylation Detection Service
Individual Nucleotide Precision: Our service excels in pinpointing 2'-O-RNA methylation modifications with single-nucleotide precision. This sophisticated approach guarantees a meticulous examination of modification locations within RNA molecules.
Efficient High-Throughput Detection: Employing advanced high-throughput techniques, we ensure the parallel and efficient identification of 2'-O-RNA modification sites throughout the entire transcriptome. This strategy allows for the simultaneous evaluation of multiple RNA molecules, furnishing a comprehensive overview of 2'-O-RNA methylation patterns.
Comprehensive Coverage Across RNA Species: Our service spans a diverse array of RNA types, encompassing mRNA, LncRNA, pri-miRNA, tRNA, and rRNA. This extensive analysis guarantees the identification of 2'-O-RNA methylation sites across various RNA molecules, contributing to a nuanced comprehension of the modification landscape.
Specialized Bioinformatic Interpretation: Supported by an adept bioinformatics team, we deliver specialized data analysis services tailored to diverse customer requirements. Our team is proficient in conducting thorough data analyses, offering insightful interpretations of results derived from 2'-O-RNA methylation detection experiments.
Sample Requirements
Sample Types: We accept various sample types, including cells, tissues, and RNA. For inquiries regarding other sample types, please contact us for further assistance.
Sample Quantities: a) Cells: 1×10^8 b) Tissues: 500 mg - 5g c) RNA: 100 μg - 300 μg (OD260/280: 1.6-2.3; RNA shows no significant degradation)
Case Study 1: Transcriptome-wide Mapping of 2'-O-Methylation on Human mRNA
This article presents, for the first time, the identification of thousands of methylation sites on human Hela and 293T cell mRNA and describes the distribution of these methylation sites. Among them, the majority of methylation sites (95.7%) are located within 2398 RefSeq annotated genes, with 95.9% occurring in protein-coding genes, and a small fraction in non-coding genes (4.1%). Within protein-coding transcripts, the data reveals that the majority of sites are located in the coding sequence (CDS) region (70.3%). Motif analysis indicates that the signature sequence for 2'-O-methylation is AGAU, followed by a high distribution of A or G bases. Furthermore, 2'-O-methylation sites are enriched in protein-coding codons for three amino acids. In summary, this study unveils, for the first time, the distribution characteristics of 2'-O-methylation in human cells.
Figure 1: Characteristics of 2'-O-RNA Methylation Sites on Human Cell mRNA Molecules
Case Study 2: Involvement of 2'-O-RNA Methyltransferase FTSJ3 in Innate Immune Regulation of HIV
Recently, the research group led by Professor Yamina Bennasser at the University of Montpellier in France discovered, for the first time, the presence of 2'-O-methylation sites on the HIV virus. These sites exhibit activity in host cell infection and are regulated by the methyltransferase FTSJ3.
FTSJ3 as a 2'-O-Methyltransferase:
Through mass spectrometry and Western blot experiments, it was observed that the methylation reader protein TRBP interacts with the methyltransferase FTSJ3, forming the TRBP-FTSJ3 complex.
Figure 2: Formation of Two Distinct Complexes by TRBP
2'-O-Methylation Modification in HIV-1 RNA Viral Particles:
Researchers employed 2'-O-methylation sequencing to examine the methylation status of HIV-1 RNA. They identified 17 2'-O-methylation sites, with 15 of them located on adenine. Subsequent methylation sequencing of HIV-1 packaged in FTSJ3-knockout cells revealed a reduction in methylation levels at multiple sites. This indicates the influence of the methyltransferase FTSJ3 on the 2'-O-methylation pattern.
Figure 3: Methylation Profile of HIV-1 RNA and the Impact of FTSJ3 Depletion
FTSJ3 Involvement in the Immune Regulation Mechanism of HIV-1:
Effect on IFN-α/IFN-β Expression: Firstly, does intracellular FTSJ3 influence the infection of host cells by the virus? The researchers used U937 monocytic cells expressing HIV RNA, with either wild-type (WT) or FTSJ3-siRNA knockdown. The results revealed a significant increase in the expression of IFN-α/IFN-β in the experimental group compared to the WT group.
Figure 4: Involvement of FTSJ3 in the Immune Regulation Process of HIV-1
MDA5 Regulation of Methyltransferase in the Immune Modulation of HIV-1:
MDA5, acting as a cytoplasmic RNA sensor, plays a crucial role in immune response. After knocking down FTSJ3, there was a decrease in IFN expression. Subsequently, upon MDA5 knockdown, IFN expression increased. This result confirms that the methyltransferase contributes to the escape of HIV RNA 2'-O-methylation from MDA5 sensing, thus completing immune evasion.
Figure 5: FTSJ3 Modulates MDA5-IFN Pathway Sensitivity
Depletion of FTSJ3 Suppresses HIV Replication:
To assess the impact of FTSJ3 on cellular immune stimulation by the virus, the authors employed FTSJ3 knockout in MDDCs during viral infection. Clearly, FTSJ3 knockout cells induced the expression of type I interferon in response to HIV virus particles, concurrently inhibiting the efficiency of HIV-1 expression. Thus, siFTSJ3-HIV non-methylated RNA induces innate immunity against viral RNA, suppressing HIV replication.
Figure 6: Depletion of FTSJ3 Suppresses HIV Replication
In summary, this study unveils, for the first time, the RNA 2'O-methylation activity of FTSJ3 and confirms that HIV virus can recruit the FTSJ3-TRBP system post-infection to accomplish 2'O-methylation modifications on viral RNA. Consequently, this modification lowers the sensitivity of the MDA5-IFN pathway, reducing immune system recognition. This novel pathway of HIV-1 innate immune evasion holds promise as a new therapeutic target for AIDS treatment.
References
- Dai, Q., Moshitch-Moshkovitz, S., Han, D. et al. Nm-seq maps 2'-O-methylation sites in human mRNA with base precision. Nat Methods 14, 695–698 (2017).
- Ringeard, M., Marchand, V., Decroly, E. et al. FTSJ3 is an RNA 2'-O-methyltransferase recruited by HIV to avoid innate immune sensing. Nature 565, 500–504 (2019).
- Motorin, Y.; Marchand, V. Detection and Analysis of RNA Ribose 2'-O-Methylations: Challenges and Solutions. Genes 2018, 9, 642.
