ALKBH1-Mediated tRNA Demethylation Regulates Translation.

tRNA is a central component of protein synthesis and the cell signaling network. One salient feature of tRNA is its heavily modified status, which can critically impact its function. Here, we show that mammalian ALKBH1 is a tRNA demethylase. It mediates the demethylation of N1-methyladenosine (m1A) in tRNAs. The ALKBH1-catalyzed demethylation of the target tRNAs results in attenuated translation initiation and decreased usage of tRNAs in protein synthesis. This process is dynamic and responds to glucose availability to affect translation. Our results uncover reversible methylation of tRNA as a new mechanism of post-transcriptional gene expression regulation.

Quantification of tRNA m1A modification by templated-ligation qPCR.

N1-methyladenosine (m1A) is a widespread modification in all eukaryotic, many archaeal, and some bacterial tRNAs. m1A is generally located in the T loop of cytosolic tRNA and between the acceptor and D stems of mitochondrial tRNAs; it is involved in the tertiary interaction that stabilizes tRNA. Human tRNA m1A levels are dynamically regulated that fine-tune translation and can also serve as biomarkers for infectious disease. Although many methods have been used to measure m1A, a PCR method to assess m1A levels quantitatively in specific tRNAs has been lacking. Here we develop a templated-ligation followed by a qPCR method (TL-qPCR) that measures m1A levels in target tRNAs. Our method uses the SplintR ligase that efficiently ligates two tRNA complementary DNA oligonucleotides using tRNA as the template, followed by qPCR using the ligation product as the template. m1A interferes with the ligation in specific ways, allowing for the quantitative assessment of m1A levels using subnanogram amounts of total RNA. We identify the features of specificity and quantitation for m1A-modified model RNAs and apply these to total RNA samples from human cells. Our method enables easy access to study the dynamics and function of this pervasive tRNA modification.

m1A regulator-mediated methylation modification patterns correlated with autophagy to predict the prognosis of hepatocellular carcinoma.

BACKGROUND: N1-methyladenosine (m1A), among the most common internal modifications on RNAs, has a crucial role to play in cancer development. The purpose of this study were systematically investigate the modification characteristics of m1A in hepatocellular carcinoma (HCC) to unveil its potential as an anticancer target and to develop a model related to m1A modification characteristics with biological functions. This model could predict the prognosis for patients with HCC. METHODS: An integrated analysis of the TCGA-LIHC database was performed to explore the gene signatures and clinical relevance of 10 m1A regulators. Furthermore, the biological pathways regulated by m1A modification patterns were investigated. The risk model was established using the genes that showed differential expression (DEGs) between various m1A modification patterns and autophagy clusters. These in vitro experiments were subsequently designed to validate the role of m1A in HCC cell growth and autophagy. Immunohistochemistry was employed to assess m1A levels and the expression of DEGs from the risk model in HCC tissues and paracancer tissues using tissue microarray. RESULTS: The risk model, constructed from five DEGs (CDK5R2, TRIM36, DCAF8L, CYP26B, and PAGE1), exhibited significant prognostic value in predicting survival rates among individuals with HCC. Moreover, HCC tissues showed decreased levels of m1A compared to paracancer tissues. Furthermore, the low m1A level group indicated a poorer clinical outcome for patients with HCC. Additionally, m1A modification may positively influence autophagy regulation, thereby inhibiting HCC cells proliferation under nutrient deficiency conditions. CONCLUSIONS: The risk model, comprising m1A regulators correlated with autophagy and constructed from five DEGs, could be instrumental in predicting HCC prognosis. The reduced level of m1A may represent a potential target for anti-HCC strategies.

The Molecular Basis of Human ALKBH3 Mediated RNA N1 -methyladenosine (m1 A) Demethylation.

N1 -methyladenosine (m1 A) is a prevalent post-transcriptional RNA modification, and the distribution and dynamics of the modification play key epitranscriptomic roles in cell development. At present, the human AlkB Fe(II)/α-ketoglutarate-dependent dioxygenase family member ALKBH3 is the only known mRNA m1 A demethylase, but its catalytic mechanism remains unclear. Here, we present the structures of ALKBH3-oligo crosslinked complexes obtained with the assistance of a synthetic antibody crystallization chaperone. Structural and biochemical results showed that ALKBH3 utilized two ß-hairpins (ß4-loop-ß5 and ß'-loop-ß'') and the α2 helix to facilitate single-stranded substrate binding. Moreover, a bubble-like region around Asp194 and a key residue inside the active pocket (Thr133) enabled specific recognition and demethylation of m1 A- and 3-methylcytidine (m3 C)-modified substrates. Mutation of Thr133 to the corresponding residue in the AlkB Fe(II)/α-ketoglutarate-dependent dioxygenase family members FTO or ALKBH5 converted ALKBH3 substrate selectivity from m1 A to N6 -methyladenosine (m6 A), as did Asp194 deletion. Our findings provide a molecular basis for understanding the mechanisms of substrate recognition and m1 A demethylation by ALKBH3. This study is expected to aid structure-guided design of chemical probes for further functional studies and therapeutic applications.

The Impact of m1A Methylation Modification Patterns on Tumor Immune Microenvironment and Prognosis in Oral Squamous Cell Carcinoma.

N1-methyladenosine (m1A) modification widely participates in the occurrence and progression of numerous diseases. Nevertheless, the potential roles of m1A in the tumor immune microenvironment (TIME) are still not fully understood. Based on 10 m1A methylation regulators, we comprehensively explored the m1A modification patterns in 502 patients with oral squamous cell carcinoma (OSCC). The m1A modification patterns were correlated with TIME characteristics and the m1A score was established to evaluate the effect of the m1A modification patterns on individual OSCC patients. The TIME characteristics and survival outcomes under the three m1A modification patterns were significantly distinct. OSCC patients in the high m1A score group were characterized by poorer prognosis, lower immune infiltration, lower ssGSEA score, lower expression levels of immune checkpoint molecules, and higher tumor mutation loads. The present study revealed that m1A modification might be associated with the TIME in OSCC, and has potential predictive ability for the prognosis of OSCC.

Analysis of RNA modifications by liquid chromatography-tandem mass spectrometry.

The analysis of RNA modifications is of high importance in order to address a wide range of biological questions. Therefore, a highly sensitive and accurate method such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) has to be available. By using different LC-MS/MS procedures, it is not only possible to quantify very low amounts of RNA modifications, but also to detect probably unknown modified nucleosides. For these cases the dynamic multiple reaction monitoring and the neutral loss scan are the most common techniques. Here, we provide the whole workflow for analyzing RNA samples regarding their modification content. This includes an equipment list, the preparation of required solutions/enzymes and the creation of an internal standard or nucleoside stocks for internal or external calibration. Furthermore, we describe the preparation of RNA samples for the subsequent LC-MS/MS analysis and the corresponding analysis process.

Landscape of N1-methyladenosin (m1A) modification pattern in colorectal cancer.

BACKGROUND: N1-methyladenosine (m1A) is a recently identified mRNA modification. However, it is still unclear that how m1A alteration affects the development of colorectal cancer (CRC). AIMS: The landscape of m1A modification patterns regarding tumor immune microenvironment (TIME) in CRC is a lack of knowledge. Thus, this study will utilize the public database to comprehensively evaluate of multiple m1A methylation regulators in CRC. METHODS AND RESULTS: We retrospectively analyzed 398 patients with CRC and 39 healthy people for negative control, using the The Cancer Genome Atlas (TCGA) database to evaluate m1A modification patterns regarding tumor immune microenvironment (TIME) in CRC. The m1Ascore was developed via principal component analysis. And its clinical value in prognosis of CRC was further explored. Our study revealed 12 key m1A-related DEGs including CLDN3, MUC2 and CCDC85B which are identified associated with invasion and metastasis in CRC. The most important biological processes linked to weak immune response and poor prognosis were the regulation of RNA metabolism and RNA biosynthesis. Furthermore, we found that compared to patients with low m1A scores, those with high m1A scores had higher percentage, larger tumor burdens, and worse prognosis. CONCLUSION: Significantly diverse m1A modification patterns can be seen in CRC. Through its impact on TIME and immunological dysfunction, the heterogeneity of m1A alteration patterns influences the prognosis of CRC. This study provided novel insights into the m1A modification in CRC which might promote the development of personalized immunotherapy strategies.

Integrated Analysis of N1-Methyladenosine Methylation Regulators-Related lncRNAs in Hepatocellular Carcinoma.

N1-methyladenosine (m1A) and long non-coding RNAs (lncRNAs) play significant roles in tumor progression in hepatocellular carcinoma (HCC). However, their association with HCC is still unclear. In this study, lncRNAs related to m1A were extracted from the mRNA expression matrix in The Cancer Genome Atlas (TCGA) database. Five m1A-related lncRNAs (AL031985.3, NRAV, WAC-AS1, AC026412.3, and AC099850.4) were identified based on lasso Cox regression and they generated a prognostic signature of HCC. The prognostic signature was identified as an independent prognosis factor in HCC patients. Moreover, the prognostic signature achieved better performance than TP53 mutation status or tumor mutational burden (TMB) scores in the stratification of patient survival. The immune landscape indicated that most immune checkpoint genes and immune cells were distributed differently between both risk groups. A higher IC50 of chemotherapeutics (sorafenib, nilotinib, sunitinib, and gefitinib) was observed in the high-risk group, and a lower IC50 of gemcitabine in the low-risk group, suggesting the potential of the prognostic signature in chemosensitivity. In addition, fifty-five potential small molecular drugs were found based on drug sensitivity and NRAV expression. Together, five m1A-related lncRNAs generated a prognostic signature that could be a promising prognostic prediction approach and therapeutic response assessment tool for HCC patients.

Sequencing methods and functional decoding of mRNA modifications.

More than 160 types of post-transcriptional RNA modifications have been reported; there is substantial variation in modification type, abundance, site, and function across species, tissues, and RNA type. The recent development of high-throughput detection technology has enabled identification of diverse dynamic and reversible RNA modifications, including N6,2'-O-dimethyladenosine (m6Am), N1-methyladenosine (m1A), 5-methylcytosine (m5C), N6-methyladenosine (m6A), pseudouridine (Ψ), and inosine (I). In this review, we focus on eukaryotic mRNA modifications. We summarize their biogenesis, regulatory mechanisms, and biological functions, as well as high-throughput methods for detection of mRNA modifications. We also discuss challenges that must be addressed in mRNA modification research.

m1A RNA Modification in Gene Expression Regulation.

N1-methyladenosine (m1A) is a prevalent and reversible post-transcriptional RNA modification that decorates tRNA, rRNA and mRNA. Recent studies based on technical advances in analytical chemistry and high-throughput sequencing methods have revealed the crucial roles of m1A RNA modification in gene regulation and biological processes. In this review, we focus on progress in the study of m1A methyltransferases, m1A demethylases and m1A-dependent RNA-binding proteins and highlight the biological mechanisms and functions of m1A RNA modification, as well as its association with human disease. We also summarize the current understanding of detection approaches for m1A RNA modification.

N1-Methyladenosine (m1A) Regulation Associated With the Pathogenesis of Abdominal Aortic Aneurysm Through YTHDF3 Modulating Macrophage Polarization.

Objectives: This study aimed to identify key AAA-related m1A RNA methylation regulators and their association with immune infiltration in AAA. Furthermore, we aimed to explore the mechanism that m1A regulators modulate the functions of certain immune cells as well as the downstream target genes, participating in the progression of AAA. Methods: Based on the gene expression profiles of the GSE47472 and GSE98278 datasets, differential expression analysis focusing on m1A regulators was performed on the combined dataset to identify differentially expressed m1A regulatory genes (DEMRGs). Additionally, CIBERSORT tool was utilized in the analysis of the immune infiltration landscape and its correlation with DEMRGs. Moreover, we validated the expression levels of DEMRGs in human AAA tissues by real-time quantitative PCR (RT-qPCR). Immunofluorescence (IF) staining was also applied in the validation of cellular localization of YTHDF3 in AAA tissues. Furthermore, we established LPS/IFN-γ induced M1 macrophages and ythdf3 knockdown macrophages in vitro, to explore the relationship between YTHDF3 and macrophage polarization. At last, RNA immunoprecipitation-sequencing (RIP-Seq) combined with PPI network analysis was used to predict the target genes of YTHDF3 in AAA progression. Results: Eight DEMRGs were identified in our study, including YTHDC1, YTHDF1-3, RRP8, TRMT61A as up-regulated genes and FTO, ALKBH1 as down-regulated genes. The immune infiltration analysis showed these DEMRGs were positively correlated with activated mast cells, plasma cells and M1 macrophages in AAA. RT-qPCR analysis also verified the up-regulated expression levels of YTHDC1, YTHDF1, and YTHDF3 in human AAA tissues. Besides, IF staining result in AAA adventitia indicated the localization of YTHDF3 in macrophages. Moreover, our in-vitro experiments found that the knockdown of ythdf3 in M0 macrophages inhibits macrophage M1 polarization but promotes macrophage M2 polarization. Eventually, 30 key AAA-related target genes of YTHDF3 were predicted, including CD44, mTOR, ITGB1, STAT3, etc. Conclusion: Our study reveals that m1A regulation is significantly associated with the pathogenesis of human AAA. The m1A "reader," YTHDF3, may participate in the modulating of macrophage polarization that promotes aortic inflammation, and influence AAA progression by regulating the expression of its target genes.

[Progress in epigenetic modification of mRNA and the function of m6A modification].

Messenger RNA (mRNA) can be modified by more than 100 chemical modifications. Among these modifications, N6-methyladenosine (m6A) is one of the most prevalent modifications. During the processes of cells differentiation, embryo development or stress, m6A can be modified on key mRNAs and regulate the progress of cells through modulating mRNA metabolism and translation. Other mRNA modifications, including N1-methyladenosine (m¹A), 5-methylcytosine (m5C) and pseudouridine, together with m6A form the epitranscriptome of mRNA that accurately modulate the mRNA translation. Here we review the types and characteristic of mRNA epigenetic modifications, especially the recent progresses of the function of m6A, we also expect the main research direction of m6A epigenetic modification in the future.

Reversible RNA Modification N1-methyladenosine (m1A) in mRNA and tRNA.

More than 100 modifications have been found in RNA. Analogous to epigenetic DNA methylation, epitranscriptomic modifications can be written, read, and erased by a complex network of proteins. Apart from N6-methyladenosine (m6A), N1-methyladenosine (m1A) has been found as a reversible modification in tRNA and mRNA. m1A occurs at positions 9, 14, and 58 of tRNA, with m1A58 being critical for tRNA stability. Other than the hundreds of m1A sites in mRNA and long non-coding RNA transcripts, transcriptome-wide mapping of m1A also identifies >20 m1A sites in mitochondrial genes. m1A in the coding region of mitochondrial transcripts can inhibit the translation of the corresponding proteins. In this review, we summarize the current understanding of m1A in mRNA and tRNA, covering high-throughput sequencing methods developed for m1A methylome, m1A-related enzymes (writers and erasers), as well as its functions in mRNA and tRNA.

High-Throughput Small RNA Sequencing Enhanced by AlkB-Facilitated RNA de-Methylation (ARM-Seq).

N 1-methyladenosine (m1A), N 3-methylcytidine (m3C), and N 1-methylguanosine (m1G) are common in transfer RNA (tRNA) and tRNA-derived fragments. These modifications alter Watson-Crick base-pairing, and cause pauses or stops during reverse transcription required for most high-throughput RNA sequencing protocols, resulting in inefficient detection of methyl-modified RNAs. Here, we describe a procedure to demethylate RNAs containing m1A, m3C, or m1G using the Escherichia coli dealkylating enzyme AlkB, along with instructions for subsequent processing with widely used protocols for small RNA sequencing.

Transcriptome-Wide Mapping of N 1-Methyladenosine Methylome.

N 1-Methyladenosine (m1A) is a prevalent posttranscriptional RNA modification and commonly found in tRNA and rRNA. Very recent works have also demonstrated the prevalence of m1A in mammalian mRNA. Hence, high-throughput methods that allow transcriptome-wide mapping of m1A will be important for further functional investigations. Here, we describe a technique called "m1A-ID-Seq", which is based on m1A immunoprecipitation and the inherent ability of m1A to stall reverse transcription, to map m1A in the transcriptome. Utilizing this technique, highly confident m1A peaks can be obtained on a transcriptome-wide level.