Post-transcriptional RNA Modification Research Articles

Abstract C010: Genes with African ancestry-specific expression in TNBC tumors also show population-specific baseline differences in normal mammary tissue

Abstract Our recent efforts to define ancestry-specific drivers in triple negative breast tumors (TNBC) has been sharply focused on identifying African (AFR) ancestry-associated gene signatures, which we have reported to be distinct from traditional approaches using SRR to categorize patients. In our AFR ancestry enriched TNBC cohort, we have identified a 613 gene signature associated with AFR ancestry, which is enriched in previously implicated canonical pathways, such as RNA post-transcriptional modification and chronic inflammation. We also reported a significant enrichment in disease/function terms associated with immune cell trafficking, where increasing African ancestry was associated with predicted activation of immune cell migration, and inhibition of immune cell activation. While these associations were determined in the TNBC tumor environment, involvement of this 613 AFR ancestry-associated gene signature has yet to be explored in the context of non-diseased breast/mammary tissue. At present, we obtained gene expression (TPM) and ancestry data from the GTEx cohort. Of 459 patients with gene expression data available for breast/mammary tissue, 47 of those were found to be of African ancestry, and 20 identified as female, and were used in subsequent analyses. We found that 17 of the 613 AFR ancestry-associated genes from TNBC tumors were also associated with AFR ancestry in non-diseased breast/mammary tissue. Interestingly we observed that some genes had differencing directionality/correlation between non-diseased and tumor tissue. Of the 17 genes, 7 were positively associated with AFR ancestry in both cohorts, including: AGMAT, CYBA, HEATR3, KCNAB2, PTGES, RAB42, and SHPK. Eight genes were found to be positively associated with AFR in normal, but negatively associated in TNBC (AC068279.3, AP001434.2, C12orf60, FNDC3B, HNRNPA1P50, RP11-214O14.1, SNORA53 and SPATA20P1). The remaining 2 were negatively associated with AFR in normal, and positively associated in tumor (AVPR2 and FAM26D). Tumor agnostic survival analysis using TCGA BRCA cohort data shows some race-specific differences in overall survival of these genes. Specifically, higher expression of AVPR2 and CYBA, and lower expression of AGMAT and SNORA53 show benefit among AA patients, but not EA patients (ns). Pathway analysis of the 17 genes shows significant enrichment with growth of mammary tumor (p = 7.3 E-07) and inflammatory response (p = 2.5 E-07) disease and function terms, specifically related to predicted activation of IFNG, AKT1, and FOS. Ancestry-specific differences in gene expression that are present in non-malignant tissue may implicate baseline differences that could play a significant role in later tumor etiology. Interestingly, 10 of 17 genes showed differing association with African ancestry, potentially revealing malignancy-induced changes to these pathways. Further analyses and curation of robust and diverse datasets of non-diseased tissue is needed to fully contextualize these findings. Citation Format: Rachel Martini, Nicolas Robine, Ishmael Kyei, Ernest Adjei, Mahteme Bekele, Olivier Elemento, Lisa Newman, Melissa Davis. Genes with African ancestry-specific expression in TNBC tumors also show population-specific baseline differences in normal mammary tissue [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C010.

Recent Advances in RNA m6A Modification in Solid Tumors and Tumor Immunity.

An analogous field to epigenetics is referred to as epitranscriptomics, which focuses on the study of post-transcriptional chemical modifications in RNA. RNA molecules, including mRNA, tRNA, rRNA, and other non-coding RNA molecules, can be edited with numerous modifications. The most prevalent modification in eukaryotic mRNA is N6-methyladenosine (m6A), which is a reversible modification found in over 7000 human genes. Recent technological advances have accelerated the characterization of these modifications, and they have been shown to play important roles in many biological processes, including pathogenic processes such as cancer. In this chapter, we discuss the role of m6A mRNA modification in cancer with a focus on solid tumor biology and immunity. m6A RNA methylation and itsregulatory proteins can play context-dependent roles in solid tumor development and progression by modulating RNA metabolism to drive oncogenic or tumor-suppressive cellular pathways. m6ARNA methylation also plays dynamic roles within both immune cells and tumor cells to mediate the anti-tumor immune response. Finally, an emerging area of research within epitranscriptomics studies the role of m6A RNA methylation in promoting sensitivity or resistance to cancer therapies, including chemotherapy, targeted therapy, and immunotherapy. Overall, our understanding of m6A RNA methylation in solid tumors has advanced significantly, and continued research is needed both to fill gaps in knowledge and to identify potential areas of focus for therapeutic development.

The m6A reader YTHDF1 attenuates fulminant hepatitis via MFG-E8 translation in an m6A dependent manner.

Background and Aims: N6-methyladenosine (m6A) is the most common post-transcriptional modification of RNA in eukaryotes, which has been demonstrated to play important roles in various biological processes. However, its roles in fulminant hepatitis remain largely unknown. In the current study, YTHDF1 expression was found to be significantly downregulated in the livers among patients, as well as murine models with fulminant hepatitis versus normal controls. Thus, we hypothesized that YTHDF1 protects against fulminant hepatitis and investigated the underlying molecular mechanisms. Methods: Fulminant hepatitis was induced by D-GalN/LPS in conventional YTHDF1 knockout (YTHDF1-/-) mice, hepatocyte-specific YTHDF1 overexpression (AAV8- YTHDF1) mice, and corresponding control mice. Primary hepatocytes were cultured and subjected to LPS insult in vitro. Hepatic histology, cell death, oxidative stress and mitochondrial function were examined to assess liver damage. The molecular mechanisms of YTHDF1 function were explored using multi-omics analysis. Results: Ablation of YTHDF1 exacerbated hepatic apoptosis and reactive oxygen species (ROS) production and increased the number of aberrant mitochondria, while YTHDF1 overexpression resulted in the opposite effects. Multiomics analysis identified MFG-E8 as the direct target of YTHDF1. YTHDF1 augmented the translation of MFG-E8 in an m6A-dependent manner without effect on its mRNA expression, thereby restoring mitochondrial function. Additionally, administration of MFG-E8 almost completely reversed the YTHDF1 deficiency-mediated exacerbation of liver injury. Conclusions: The current study suggested that the m6A reader YTHDF1 alleviates cell death, enhances antioxidant capacity and restores mitochondrial function in fulminant hepatitis by promoting MFG-E8 protein translation in an m6A-dependent manner.

SnoRNAs are Deregulated in Patients with Parkinson’s Disease

Purpose: Small nucleolar RNAs are ranging from 65 to 300 nucleotides in length that mediate post-transcriptional RNA modifications. They don’t have a 5′-Cap and a poly-A tail and are categorized as C/D box snoRNAs, H/ACA box snoRNAs, and small Cajal body-specific RNAs. snoRNAs have essential roles in important biological processes such as transcription, RNA splicing, cell cycle, and etc. In this study, we tried to reveal differential expressions of snoRNAs in PBMCs of patients with Parkinson’s Disease by microarray analysis.
 Materials and methods: Patients (n=3) who are considered to have a unilateral onset history and a good response to dopaminergic treatment in the first years were included in the study. 10 ml peripheral blood sample was taken for peripheral blood mononuclear cell isolation. Total RNA was extracted using GeneAll® Hybrid-R™ kit and microarray analysis was performed by using Affymetrix GeneChip Human ST 2.0 platform. Raw data were extracted using Affymetrix Command Console Software 1.1. KEGG pathway and GO terms analyses were performed and protein-protein interaction of host genes were determined by using STRING database.
 Results: Data from patients revealed that there were 28 snoRNAs were downregulated and 3 snoRNAs were upregulated. 
 Conclusion: Here in this study, we evaluated the differential expressions of snoRNAs in patients with a definitive diagnosis of PD by microarray analysis and observed deregulated expressions of some snoRNAs. Differential expression of snoRNA may cause changes in the transcriptional activity of host genes and thus can serve as biomarkers for diseases.

Transcriptome-wide analysis of pseudouridylation in Drosophila melanogaster.

Pseudouridine (Psi) is one of the most frequent post-transcriptional modification of RNA. Enzymatic Psi modification occurs on rRNA, snRNA, snoRNA, tRNA, and non-coding RNA and has recently been discovered on mRNA. Transcriptome-wide detection of Psi (Psi-seq) has yet to be performed for the widely studied model organism Drosophila melanogaster. Here, we optimized Psi-seq analysis for this species and have identified thousands of Psi modifications throughout the female fly head transcriptome. We find that Psi is widespread on both cellular and mitochondrial rRNAs. In addition, more than a thousand Psi sites were found on mRNAs. When pseudouridylated, mRNAs frequently had many Psi sites. Many mRNA Psi sites are present in genes encoding for ribosomal proteins, and many are found in mitochondrial encoded RNAs, further implicating the importance of pseudouridylation for ribosome and mitochondrial function. The 7SLRNA of the signal recognition particle is the non-coding RNA most enriched for Psi. The 3 mRNAs most enriched for Psi encode highly expressed yolk proteins (Yp1, Yp2, and Yp3). By comparing the pseudouridine profiles in the RluA-2 mutant and the w1118 control genotype, we identified Psi sites that were missing in the mutant RNA as potential RluA-2 targets. Finally, differential gene expression analysis of the mutant transcriptome indicates a major impact of loss of RluA-2 on the ribosome and translational machinery.

M6A demethylase FTO regulate CTNNB1 to promote adipogenesis of chicken preadipocyte

BackgroundN6-methyladenosine (m6A) is an abundant post-transcriptional RNA modification that affects various biological processes. The fat mass and obesity-associated (FTO) protein, a demethylase encoded by the FTO gene, has been found to regulate adipocyte development in an m6A-dependent manner in multiple species. However, the effects of the m6A methylation and FTO demethylation functions on chicken adipogenesis remain unclear. This study aims to explore the association between m6A modification and chicken adipogenesis and the underlying mechanism by which FTO affects chicken preadipocyte development.ResultsThe association between m6A modification and chicken lipogenesis was assessed by treating chicken preadipocytes with different doses of methyl donor betaine and methylation inhibitor cycloleucine. The results showed that betaine significantly increased methylation levels and inhibited lipogenesis, and the inverse effect was found in preadipocytes after cycloleucine treatment. Overexpression of FTO significantly inhibited m6A levels and promoted proliferation and differentiation of chicken preadipocytes. Silencing FTO showed opposite results. Mechanistically, FTO overexpression increased the expression of catenin beta 1 (CTNNB1) by improving RNA stability in an m6A-dependent manner, and we proved that FTO could directly target CTNNB1. Furthermore, CTNNB1 may be a positive regulator of adipogenesis in chicken preadipocytes.Conclusionsm6A methylation of RNA was negatively associated with adipogenesis of chicken preadipocytes. FTO could regulate CTNNB1 expression in a demethylation manner to promote lipogenesis.

N6-methyladenosine (m6A) RNA modification in the pathophysiology of heart failure: a narrative review.

Heart failure is the end-stage of various cardiovascular diseases. Recent progress in molecular biology has facilitated the understanding of the mechanisms of heart failure development at the molecular level. N6-adenosine methylation (m6A) is a post-transcriptional modification of RNA. Recent research work reported that m6A regulates gene expression and subsequently affects the activation of cell signaling pathways related to heart failure. Moreover, m6A regulators like methyltransferase-like 3 (METTL3) were reported to participate in myocardium hypertrophy. However, the current research work related to the role of m6A participating in the occurrence of heart failure is rare in some aspects like immune cell infiltration and diabetic heart diseases. Thus, it is reasonable to review the current achievements and provide further study orientation. We searched related literature using the keywords: m6A AND heart failure in PubMed, Web of Science and Medline. The language was confined to English. The published year of searched literature ranged from 2012 to 2022. The searched results were put into Endnote software for management. Two authors investigated the searching terms and reviewed the full text of selected terms. m6A and its regulators are involved in the metabolism of various types of RNAs. m6A modification can regulate various types of cell signaling pathways related to the heart failure via interaction with m6A regulators. m6A and its regulators broadly participate in the myocardium fibrosis, myocardium hypertrophy, myocardial cell apoptosis, and ischemic reperfusion injury. Specifically, m6A participates in the cell apoptosis via regulation of autophagy flux. However, the current research work does not have enough evidence to prove that m6A regulator played its specific effect on the target transcript via regulating the m6A level. m6A and its regulators participates in the progression of heart failure via modifying the RNA level. Future investigation of m6A should focus on the interaction between the m6A regulators and targeted transcript. Besides, the regulation role of m6A in immune cell infiltration and diabetic heart diseases should also be focused.

Integrated analysis from multi-center studies identities m7G-derived modification pattern and risk stratification system in skin cutaneous melanoma.

The m7G modification has been proven to play an important role in RNA post-transcriptional modification and protein translation. However, the potential role of m7G modification patterns in assessing the prognosis of Skin cutaneous melanoma (SKCM) and tumor microenvironment (TME) has not been well studied. In this study, we investigated and finally identified 21 available m7G-related genes. We used hierarchical clustering (K-means) to classify 743 SKCM patients into three m7G-modified subtypes named m7G/gene cluster-A, B, C. We found that both m7G cluster B and gene cluster B exhibited higher prognosis and higher immune cell infiltration in TME compared to other subtypes. EIF4E3 and IFIT5, two m7G related genes, were both markedly elevated in Cluster B. Then, we constructed an m7G score system utilizing principal component analysis (PCA) in order to evaluate the patients' prognosis. High m7G score subtype was associated with better survival prognosis and active immune response. Overall, this article revealed that m7G modification patterns were involved in the development of the tumor microenvironment. Evaluating patients' m7G modification patterns will enhance our understanding of TME characteristics and help to guide personal treatment in clinics in the future.

Abstract IA008: Chromatin regulation by reversible chromatin-associated RNA methylation

Abstract Over 150 types of post-transcriptional RNA modifications have been identified in all kingdoms of life. We have previously discovered two RNA demethylases, FTO and ALKBH5, which catalyze oxidative demethylation of the most prevalent modifications of mammalian messenger RNA (mRNA) and other nuclear RNA, N6-methyladenosine (m6A). We have also characterized reader proteins that preferentially recognize methylated transcripts to facilitate either decay or translation of these target RNAs. Most recently we have discovered m6A methylation of chromatin-associated regulatory RNAs (carRNAs), which modulates chromatin state and transcription in mouse embryonic stem cells (mESCs). These findings suggest that reversible RNA modification could impact chromatin regulation analogous to the reversible DNA and histone chemical modifications. I will present recent mechanistic investigation of chromatin and transcription regulation through m6A methylation and demethylation, and impacts on mammalian development and cancer progression. Citation Format: Chuan He. Chromatin regulation by reversible chromatin-associated RNA methylation. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr IA008.

Targeting FTO Suppresses Pancreatic Carcinogenesis via Regulating Stem Cell Maintenance and EMT Pathway

N6-methyladenosine (m6A) is the most prevalent post-transcriptional RNA modification regulating cancer self-renewal. However, despite its functional importance and prognostic implication in tumorigenesis, the relevance of FTO, an m6A eraser, in pancreatic cancer (PC) remains elusive. Here, we establish the oncogenic role played by FTO overexpression in PC. FTO is upregulated in PC cells compared to normal human pancreatic ductal epithelial (HPDE) cells. Both RNAi depletion and CS1-mediated pharmacological inhibition of FTO caused a diminution of PC cell proliferation via cell cycle arrest in the G1 phase and p21cip1 and p27kip1 induction. While HPDE cells remain insensitive to CS1 treatment, FTO overexpression confers enhancements in growth, motility, and EMT transition, thereby inculcating tumorigenic properties in HPDE cells. Notably, shRNA-mediated FTO depletion in PC cells impairs their mobility and invasiveness, leading to EMT reversal. Mechanistically, this was associated with impaired tumorsphere formation and reduced expression of CSCs markers. Furthermore, FTO depletion in PC cells weakened their tumor-forming capabilities in nude mice; those tumors had increased apoptosis, decreased proliferation markers, and MET conversion. Collectively, our study demonstrates the functional importance of FTO in PC and the maintenance of CSCs via EMT regulation. Thus, FTO may represent an attractive therapeutic target for PC.

Identification of putative reader proteins of 5-methylcytosine and its derivatives in Caenorhabditis elegans RNA.

Background: Methylation of carbon-5 of cytosines (m 5C) is a conserved post-transcriptional nucleotide modification of RNA with widespread distribution across organisms. It can be further modified to yield5-hydroxymethylcytidine (hm 5C), 5-formylcytidine (f 5C), 2´-O-methyl-5-hydroxymethylcytidine (hm 5Cm) and 2´-O-methyl-5-formylcytidine (f 5Cm).How m 5C, and specially its derivates, contribute to biology mechanistically is poorly understood. We recently showed that m 5C is required for Caenorhabditis elegans development and fertility under heat stress. m 5C has been shown to participate in mRNA transport and maintain mRNA stability through its recognition by the reader proteins ALYREF and YBX1, respectively. Hence, identifying readers for RNA modifications can enhance our understanding in the biological roles of these modifications. Methods: To contribute to the understanding of how m 5C and its oxidative derivatives mediate their functions, we developed RNA baits bearing modified cytosines in diverse structural contexts to pulldown potential readers in C. elegans. Potential readers were identified using mass spectrometry. The interaction of two of the putative readers with m 5C was validated using immunoblotting. Results: Our mass spectrometry analyses revealed unique binding proteins for each of the modifications. In silico analysis for phenotype enrichments suggested that hm 5Cm unique readers are enriched in proteins involved in RNA processing, while readers for m 5C, hm 5C and f 5C are involved in germline processes. We validated our dataset by demonstrating that the nematode ALYREF homologues ALY-1 and ALY-2 preferentially bind m 5C in vitro. Finally, sequence alignment analysis showed that several of the putative m 5C readers contain the conserved RNA recognition motif (RRM), including ALY-1 and ALY-2. Conclusions: The dataset presented here serves as an important scientific resource that will support the discovery of new functions of m 5C and its derivatives. Furthermore, we demonstrate that ALY-1 and ALY-2 bind to m 5C in C. elegans.

DirectRMDB: a database of post-transcriptional RNA modifications unveiled from direct RNA sequencing technology.

With advanced technologies to map RNA modifications, our understanding of them has been revolutionized, and they are seen to be far more widespread and important than previously thought. Current next-generation sequencing (NGS)-based modification profiling methods are blind to RNA modifications and thus require selective chemical treatment or antibody immunoprecipitation methods for particular modification types. They also face the problem of short read length, isoform ambiguities, biases and artifacts. Direct RNA sequencing (DRS) technologies, commercialized by Oxford Nanopore Technologies (ONT), enable the direct interrogation of any given modification present in individual transcripts and promise to address the limitations of previous NGS-based methods. Here, we present the first ONT-based database of quantitative RNA modification profiles, DirectRMDB, which includes 16 types of modification and a total of 904,712 modification sites in 25 species identified from 39 independent studies. In addition to standard functions adopted by existing databases, such as gene annotations and post-transcriptional association analysis, we provide a fresh view of RNA modifications, which enables exploration of the epitranscriptome in an isoform-specific manner. The DirectRMDB database is freely available at: http://www.rnamd.org/directRMDB/.

The RNA demethylase ALKBH5 promotes the progression and angiogenesis of lung cancer by regulating the stability of the LncRNA PVT1

BackgroundN6-methyladenosine (m6A) is the most common posttranscriptional modification of RNA and plays critical roles in human cancer progression. However, the biological function of m6A methylation requires further studied in cancer, especially in tumor angiogenesis.MethodsA public database was used to analyze the expression and overall survival of ALKBH5 and PVT1 in lung cancer patients. CCK-8 and colony formation assays were performed to detect cell proliferation, a transwell assay was used to assess cell migration, and a tube formation assay was performed to assess angiogenic potential in vitro. A zebrafish lung cancer xenograft model was used to verify the function of ALKBH5 and PVT1 in vivo. Western blot assays were used to measure the relative protein expression in lung cancer cells. SRAMP predictor analysis and RNA stability experiments were used to examine the potential m6A modification.ResultsBioinformatics analysis showed that the expression levels of m6A-related genes were changed significantly in lung cancer tissues compared with normal lung tissues. We then identified that ALKBH5 was upregulated in lung cancer tissues and associated with poor prognosis of lung cancer patients by analyzing a public database. Knockdown of ALKBH5 inhibited the proliferation and migration of cultured lung cancer cell lines. Zebrafish lung cancer xenografts showed that ALKBH5 silencing also suppressed the growth and metastasis of lung cancer cells. Moreover, knockdown of ALKBH5 inhibited the angiogenesis of lung cancer in vitro and in vivo. Mechanistic studies showed that knockdown of ALKBH5 decreased the expression and stability of PVT1 in lung cancer cells. We next observed that PVT1 promoted the progression of lung cancer cells in vitro and in vivo and regulated the expression of VEGFA and angiogenesis in lung cancer. Finally, rescue experiments revealed that ALKBH5 regulated the proliferation, migration and angiogenesis of lung cancer cells, partially through PVT1.ConclusionOur results demonstrate that ALKBH5 promotes the progression and angiogenesis of lung cancer by regulating the expression and stability of PVT1, which provides a potential prognostic and therapeutic target for lung cancer patients.

Transcriptome-wide analysis of m6A methylation reveals genetic responses to cadmium stress at germination stage in rice

Excess cadmium (Cd) seriously suppresses plant growth and development. Rice (Oryza sativa L.) is more susceptible to Cd toxicity than other crops. N6-methyladenosine (m6A) modifications play important regulatory roles in plant adaption to various stresses. However, the post-transcriptional RNA modification in response to Cd stress is still largely unclear in rice. In this study, seeds of the rice cultivar Nipponbare were treated with 0.5 μM CdCl2 for 10 days at germination stage, and then the roots were sampled for the transcriptome-m6A methylation profiling. Here, 1920 differential peaks (DPs) of m6A modifications and 1710 differentially expressed genes (DEGs) were identified in the roots after Cd treatment. Among them, root cell expansion suppressor OsRePRPs were remarkably up-regulated, while auxin/cytokinin-related genes were differentially modified by m6A under Cd stress including OsYUCCA1, OsCKX4, OsRR6, and OsEIL1. Besides, peroxidases (PRXs), metallothioneins (MTs), glutathione S-transferases (GSTs) and sulfate transporter SULTRs were modified by m6A and significantly up-regulated, which might be related to the Cd detoxification via the cell wall modifications and Cd chelate formation. Moreover, several m6A modified transcription factors and MAPK signaling genes showed obviously increased expression, consequently participating in the Cd-response regulatory network as the upstream components. The present study proposes a working model for the m6A modification and transcriptional regulation in rice during germination, and also provides new insights into how plants alleviate Cd toxicity.

Spatiotemporal and genetic regulation of A-to-I editing throughout human brain development.

Posttranscriptional RNA modifications by adenosine-to-inosine (A-to-I) editing are abundant in the brain, yet elucidating functional sites remains challenging. To bridge this gap, we investigate spatiotemporal and genetically regulated A-to-I editing sites across prenatal and postnatal stages of human brain development. More than 10,000 spatiotemporally regulated A-to-I sites were identified that occur predominately in 3' UTRs and introns, as well as 37 sites that recode amino acids in protein coding regions with precise changes in editing levels across development. Hyper-edited transcripts are also enriched in the aging brain and stabilize RNA secondary structures. These features are conserved in murine and non-human primate models of neurodevelopment. Finally, thousands of cis-editing quantitative trait loci (edQTLs) were identified with unique regulatory effects during prenatal and postnatal development. Collectively, this work offers a resolved atlas linking spatiotemporal variation in editing levels to genetic regulatory effects throughout distinct stages of brain maturation.

Prediction and Motif Analysis of 2’-O-methylation Using a Hybrid Deep Learning Model from RNA Primary Sequence and Nanopore Signals

Background: 2’-O-Methylation (2’-O-Me) is a post-transcriptional RNA modification that occurs in the ribose sugar moiety of all four nucleotides and is abundant in both coding and non-coding RNAs. Accurate prediction of each subtype of 2’-O-Me (Am, Cm, Gm, Um) helps understand their role in RNA metabolism and function. Objective: This study aims to build models that can predict each subtype of 2’-O-Me from RNA sequence and nanopore signals and exploit the model interpretability for sequence motif mining. Methods: We first propose a novel deep learning model DeepNm to better capture the sequence features of each subtype with a multi-scale framework. Based on DeepNm, we continue to propose HybridNm, which combines sequences and nanopore signals through a dual-path framework. The nanopore signalderived features are first passed through a convolutional layer and then merged with sequence features extracted from different scales for final classification. Results: A 5-fold cross-validation process on Nm-seq data shows that DeepNm outperforms two stateof- the-art 2’-O-Me predictors. After incorporating nanopore signal-derived features, HybridNm further achieved significant improvements. Through model interpretation, we identified not only subtypespecific motifs but also revealed shared motifs between subtypes. In addition, Cm, Gm, and Um shared motifs with the well-studied m6A RNA methylation, suggesting a potential interplay among different RNA modifications and the complex nature of epitranscriptome regulation. Conclusion: The proposed frameworks can be useful tools to predict 2’-O-Me subtypes accurately and reveal specific sequence patterns.

RNA m 6A Modification and Its Roles in Immune Function Regulation

N 6-methyladenosine (m 6A) is the most prevalent post-transcriptional RNA modification in eukaryotic cells. m 6A methyltransferase complexes and demethylases are involved in the dynamic and reversible regulation of m 6A modification, while m 6A binding proteins could specifically identify m 6A modification and regulate RNA metabolism, including splicing, translocation, stability, and translation. m 6A is found to play a vital role in all the fundamental biological processes, including the regulation of the immune system, from normal development to diseases. The normal development and functioning of the immune system rely on the precise regulation of the relevant gene expression, and m 6A modification is crucial to the regulation. Herein, we reviewed the concept, the functions, and the mechanism of m 6A modification, concentrating on its role in immune system function modulation. Furthermore, we highlighted present obstacles and future research directions in the field, intending to provide a theoretical foundation and reference for future study on how m 6A modification is involved in immune regulation.

DHU-Pred: accurate prediction of dihydrouridine sites using position and composition variant features on diverse classifiers.

BackgroundDihydrouridine (D) is a modified transfer RNA post-transcriptional modification (PTM) that occurs abundantly in bacteria, eukaryotes, and archaea. The D modification assists in the stability and conformational flexibility of tRNA. The D modification is also responsible for pulmonary carcinogenesis in humans.ObjectiveFor the detection of D sites, mass spectrometry and site-directed mutagenesis have been developed. However, both are labor-intensive and time-consuming methods. The availability of sequence data has provided the opportunity to build computational models for enhancing the identification of D sites. Based on the sequence data, the DHU-Pred model was proposed in this study to find possible D sites.MethodologyThe model was built by employing comprehensive machine learning and feature extraction approaches. It was then validated using in-demand evaluation metrics and rigorous experimentation and testing approaches.ResultsThe DHU-Pred revealed an accuracy score of 96.9%, which was considerably higher compared to the existing D site predictors.Availability and ImplementationA user-friendly web server for the proposed model was also developed and is freely available for the researchers.

Reduced N6-Methyladenosine Mediated by METTL3 Acetylation Promotes MTF1 Expression and Hepatocellular Carcinoma Cell Growth.

N6-Methyladenosine (m6A), one of the post-transcriptional modifications of RNA, is important in hepatocellular carcinoma (HCC). However, the mechanism of its regulation remains elusive. We here show that exposure of HCC cells to sulfatide significantly reduced the total mRNA m6A modification. Interestingly, METTL3 protein was robustly acetylated and the binding of METTL3 to MTF1 mRNA, METTL14 or WTAP was weakened in cells treated with sulfatide. Further investigation of the METTL3 complex revealed recruitment of the deacetylase scaffold SIN3B, but a diminished level of histone deacetylase HDAC2, which might enhance the acetylation of METTL3. The m6A abundance in MTF1 mRNA was markedly decreased in cells after sulfatide treatment. The expression of MTF1, a zinc-dependent transcription factor, was significantly strengthened with reduced m6A modification. Sulfatide prolonged the half-life of MTF1 mRNA, while the mutation (A to C) on 7 methylation sites in the 3'UTR of MTF1 mRNA enhanced MTF1 mRNA stability. 3-deaza-adenosine, an m6A methylation inhibitor, significantly reduced the m6A modification of MTF1 mRNA but extended its half-life time. Importantly, overexpression of MTF1 prompted HCC cell proliferation and was associated with poor prognosis. In conclusion, the METTL3-METTL14-WTAP complex was regulated by acetylation induced by sulfatide to control MTF1 m6A methylation and its mRNA transcription, which was important for the tumor growth and migration of HCC.

Occurrence of RNA post-transcriptional modifications in plant viruses and viroids and their correlation with structural and functional features

Post-transcriptional modifications of RNA bases are widespread across all the tree of life and have been linked to RNA maturation, stability, and molecular interactions. RNA modifications have been extensively described in endogenous eukaryotic mRNAs, however, little is known about the presence of RNA modifications in plant viral and subviral RNAs. Here, we used a computational approach to infer RNA modifications in plant-pathogenic viruses and viroids using high-throughput annotation of modified ribonucleotides (HAMR), a software that predicts modified ribonucleotides using high-throughput RNA sequencing data. We analyzed datasets from representative members of different plant viruses and viroids and compared them to plant-endogenous mRNAs. Our approach was able to predict potential RNA chemical modifications (RCMs) in all analyzed pathogens. We found that both DNA and RNA viruses presented a wide range of RCM proportions while viroids had lowest values. Furthermore, we found that for viruses with segmented genomes, some genomic RNAs had a higher proportion of RCM. Interestingly, nuclear-replicating viroids showed most of the predicted modifications located in the pathogenesis region, pointing towards a possible functional role of RCMs in their infectious cycle. Thus, our results strongly suggest that plant viral and subviral RNAs might contain a variety of previously unreported RNA modifications, thus opening a new perspective in the multifaceted process of plant-pathogen interactions.