ABSTRACT
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.
Subject(s)
AlkB Homolog 1, Histone H2a Dioxygenase/metabolism , Gene Expression Regulation , Protein Biosynthesis/genetics , RNA, Transfer/metabolism , Adenosine/analogs & derivatives , Adenosine/metabolism , AlkB Homolog 1, Histone H2a Dioxygenase/genetics , Glucose/deficiency , HeLa Cells , Humans , Methylation , Polyribosomes/metabolismABSTRACT
N6-methyladenosine (m6A) of mRNAs modulated by the METTL3-METTL14-WTAP-RBM15 methyltransferase complex and m6A demethylases such as FTO play important roles in regulating mRNA stability, splicing, and translation. Here, we demonstrate that FTO-IT1 long noncoding RNA (lncRNA) was upregulated and positively correlated with poor survival of patients with wild-type p53-expressing prostate cancer (PCa). m6A RIP-seq analysis revealed that FTO-IT1 knockout increased mRNA m6A methylation of a subset of p53 transcriptional target genes (e.g., FAS, TP53INP1, and SESN2) and induced PCa cell cycle arrest and apoptosis. We further showed that FTO-IT1 directly binds RBM15 and inhibits RBM15 binding, m6A methylation, and stability of p53 target mRNAs. Therapeutic depletion of FTO-IT1 restored mRNA m6A level and expression of p53 target genes and inhibited PCa growth in mice. Our study identifies FTO-IT1 lncRNA as a bona fide suppressor of the m6A methyltransferase complex and p53 tumor suppression signaling and nominates FTO-IT1 as a potential therapeutic target of cancer.
Subject(s)
Neoplasms , RNA, Long Noncoding , Male , Mice , Animals , RNA, Long Noncoding/genetics , Tumor Suppressor Protein p53/genetics , Adenosine/metabolism , RNA, Messenger/genetics , Methyltransferases/genetics , Methyltransferases/metabolism , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/metabolismABSTRACT
RNA-binding proteins (RBPs) control messenger RNA fate in neurons. Here, we report a mechanism that the stimuli-induced neuronal translation is mediated by phosphorylation of a YTHDF1-binding protein FMRP. Mechanistically, YTHDF1 can condense with ribosomal proteins to promote the translation of its mRNA targets. FMRP regulates this process by sequestering YTHDF1 away from the ribosome; upon neuronal stimulation, FMRP becomes phosphorylated and releases YTHDF1 for translation upregulation. We show that a new small molecule inhibitor of YTHDF1 can reverse fragile X syndrome (FXS) developmental defects associated with FMRP deficiency in an organoid model. Our study thus reveals that FMRP and its phosphorylation are important regulators of activity-dependent translation during neuronal development and stimulation and identifies YTHDF1 as a potential therapeutic target for FXS in which developmental defects caused by FMRP depletion could be reversed through YTHDF1 inhibition.
Subject(s)
Fragile X Mental Retardation Protein , Fragile X Syndrome , Humans , Phosphorylation , Fragile X Mental Retardation Protein/genetics , Fragile X Mental Retardation Protein/metabolism , Neurons/metabolism , Fragile X Syndrome/genetics , Fragile X Syndrome/metabolism , Ribosomal Proteins/metabolism , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolismABSTRACT
Cellular RNAs are naturally decorated with a variety of chemical modifications. The structural diversity of the modified nucleosides provides regulatory potential to sort groups of RNAs for organized metabolism and functions, thus affecting gene expression. Recent years have witnessed a burst of interest in and understanding of RNA modification biology, thanks to the emerging transcriptome-wide sequencing methods for mapping modified sites, highly sensitive mass spectrometry for precise modification detection and quantification, and extensive characterization of the modification "effectors," including enzymes ("writers" and "erasers") that alter the modification level and binding proteins ("readers") that recognize the chemical marks. However, challenges remain due to the vast heterogeneity in expression abundance of different RNA species, further complicated by divergent cell-type-specific and tissue-specific expression and localization of the effectors as well as modifications. In this review, we highlight recent progress in understanding the function of N6-methyladenosine (m6A), the most abundant internal mark on eukaryotic mRNA, in light of the specific biological contexts of m6A effectors. We emphasize the importance of context for RNA modification regulation and function.
Subject(s)
Adenosine/analogs & derivatives , Methylation , RNA, Messenger/genetics , RNA/genetics , Adenosine/genetics , Eukaryotic Cells/metabolism , Gene Expression Regulation/genetics , Organ Specificity/genetics , RNA Processing, Post-Transcriptional/genetics , TranscriptomeABSTRACT
FTO, the first RNA demethylase discovered, mediates the demethylation of internal N6-methyladenosine (m6A) and N6, 2-O-dimethyladenosine (m6Am) at the +1 position from the 5' cap in mRNA. Here we demonstrate that the cellular distribution of FTO is distinct among different cell lines, affecting the access of FTO to different RNA substrates. We find that FTO binds multiple RNA species, including mRNA, snRNA, and tRNA, and can demethylate internal m6A and cap m6Am in mRNA, internal m6A in U6 RNA, internal and cap m6Am in snRNAs, and N1-methyladenosine (m1A) in tRNA. FTO-mediated demethylation has a greater effect on the transcript levels of mRNAs possessing internal m6A than the ones with cap m6Am in the tested cells. We also show that FTO can directly repress translation by catalyzing m1A tRNA demethylation. Collectively, FTO-mediated RNA demethylation occurs to m6A and m6Am in mRNA and snRNA as well as m1A in tRNA.
Subject(s)
Adenosine/analogs & derivatives , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/physiology , 3T3-L1 Cells , Adenosine/metabolism , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/metabolism , Animals , Cell Nucleus , Cytoplasm , Demethylation , Gene Expression/genetics , HEK293 Cells , HeLa Cells , Humans , Methylation , Mice , RNA Processing, Post-Transcriptional/physiology , RNA, Messenger/metabolism , RNA, Small Nuclear/metabolism , RNA, Transfer/metabolismABSTRACT
Global genome repair (GGR), a subpathway of nucleotide excision repair, corrects bulky helix-distorting DNA lesions across the whole genome and is essential for preventing mutagenesis and skin cancer. Here, we show that METTL14 (methyltransferase-like 14), a critical component of the N6-methyladenosine (m6A) RNA methyltransferase complex, promotes GGR through regulating m6A mRNA methylation-mediated DDB2 translation and suppresses ultraviolet B (UVB) radiation-induced skin tumorigenesis. UVB irradiation down-regulates METTL14 protein through NBR1-dependent selective autophagy. METTL14 knockdown decreases GGR and DDB2 abundance. Conversely, overexpression of wild-type METTL14 but not its enzymatically inactive mutant increases GGR and DDB2 abundance. METTL14 knockdown decreases m6A methylation and translation of the DDB2 transcripts. Adding DDB2 reverses the GGR repair defect in METTL14 knockdown cells, indicating that METTL14 facilitates GGR through regulating DDB2 m6A methylation and translation. Similarly, knockdown of YTHDF1, an m6A reader promoting translation of m6A-modified transcripts, decreases DDB2 protein levels. Both METTL14 and YTHDF1 bind to the DDB2 transcript. In mice, skin-specific heterozygous METTL14 deletion increases UVB-induced skin tumorigenesis. Furthermore, METTL14 as well as DDB2 is down-regulated in human and mouse skin tumors and by chronic UVB irradiation in mouse skin, and METTL14 level is associated with the DDB2 level, suggesting a tumor-suppressive role of METTL14 in UVB-associated skin tumorigenesis in association with DDB2 regulation. Taken together, these findings demonstrate that METTL14 is a target for selective autophagy and acts as a critical epitranscriptomic mechanism to regulate GGR and suppress UVB-induced skin tumorigenesis.
Subject(s)
Carcinogenesis/genetics , DNA Repair/physiology , Methyltransferases/physiology , Skin Neoplasms/genetics , Animals , Autophagy , Cell Line, Tumor , DNA Damage , DNA Repair/genetics , DNA-Binding Proteins/metabolism , Female , Genes, Tumor Suppressor/radiation effects , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Male , Membrane Glycoproteins/metabolism , Methylation , Methyltransferases/genetics , Mice , Nerve Tissue Proteins/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Skin Neoplasms/etiology , Ultraviolet RaysABSTRACT
Human respiratory syncytial virus (RSV) is the leading cause of respiratory tract infections in humans. A well-known challenge in the development of a live attenuated RSV vaccine is that interferon (IFN)-mediated antiviral responses are strongly suppressed by RSV nonstructural proteins which, in turn, dampens the subsequent adaptive immune responses. Here, we discovered a novel strategy to enhance innate and adaptive immunity to RSV infection. Specifically, we found that recombinant RSVs deficient in viral RNA N6-methyladenosine (m6A) and RSV grown in m6A methyltransferase (METTL3)-knockdown cells induce higher expression of RIG-I, bind more efficiently to RIG-I, and enhance RIG-I ubiquitination and IRF3 phosphorylation compared to wild-type virion RNA, leading to enhanced type I IFN production. Importantly, these m6A-deficient RSV mutants also induce a stronger IFN response in vivo, are significantly attenuated, induce higher neutralizing antibody and T cell immune responses in mice and provide complete protection against RSV challenge in cotton rats. Collectively, our results demonstrate that inhibition of RSV RNA m6A methylation enhances innate immune responses which in turn promote adaptive immunity.
Subject(s)
Adenosine/analogs & derivatives , RNA, Viral , Respiratory Syncytial Virus Infections/immunology , Respiratory Syncytial Virus, Human/immunology , Adaptive Immunity , Animals , Immunity, Innate , Methylation , Mice , RatsABSTRACT
YTHDC1 has distinct functions as a nuclear N6-methyladenosine (m6A) reader in regulating RNA metabolism. Here we show that YTHDC1 is overexpressed in acute myeloid leukemia (AML) and that it is required for the proliferation and survival of human AML cells. Genetic deletion of Ythdc1 markedly blocks AML development and maintenance as well as self-renewal of leukemia stem cells (LSCs) in vivo in mice. We found that Ythdc1 is also required for normal hematopoiesis and hematopoietic stem and progenitor cell (HSPC) maintenance in vivo. Notably, Ythdc1 haploinsufficiency reduces self-renewal of LSCs but not HSPCs in vivo. YTHDC1 knockdown has a strong inhibitory effect on proliferation of primary AML cells. Mechanistically, YTHDC1 regulates leukemogenesis through MCM4, which is a critical regulator of DNA replication. Our study provides compelling evidence that shows an oncogenic role and a distinct mechanism of YTHDC1 in AML.
Subject(s)
Gene Expression Regulation, Leukemic , Leukemia, Myeloid, Acute/genetics , Minichromosome Maintenance Proteins/genetics , Nerve Tissue Proteins/genetics , RNA Splicing Factors/genetics , Adenosine/analogs & derivatives , Adenosine/genetics , Animals , Carcinogenesis/genetics , Cell Line, Tumor , DNA Replication , Humans , Mice, Transgenic , Minichromosome Maintenance Complex Component 4/genetics , Up-RegulationABSTRACT
Faithful genome integrity maintenance plays an essential role in cell survival. Here, we identify the RNA demethylase ALKBH5 as a key regulator that protects cells from DNA damage and apoptosis during reactive oxygen species (ROS)-induced stress. We find that ROS significantly induces global mRNA N6-methyladenosine (m6A) levels by modulating ALKBH5 post-translational modifications (PTMs), leading to the rapid and efficient induction of thousands of genes involved in a variety of biological processes including DNA damage repair. Mechanistically, ROS promotes ALKBH5 SUMOylation through activating ERK/JNK signaling, leading to inhibition of ALKBH5 m6A demethylase activity by blocking substrate accessibility. Moreover, ERK/JNK/ALKBH5-PTMs/m6A axis is activated by ROS in hematopoietic stem/progenitor cells (HSPCs) in vivo in mice, suggesting a physiological role of this molecular pathway in the maintenance of genome stability in HSPCs. Together, our study uncovers a molecular mechanism involving ALKBH5 PTMs and increased mRNA m6A levels that protect genomic integrity of cells in response to ROS.
Subject(s)
AlkB Homolog 5, RNA Demethylase/metabolism , DNA Damage , DNA Repair , Reactive Oxygen Species/metabolism , AlkB Homolog 5, RNA Demethylase/genetics , Animals , Apoptosis/drug effects , Apoptosis/genetics , Bone Marrow Cells/drug effects , Bone Marrow Cells/metabolism , Cell Line, Tumor , DNA Damage/drug effects , DNA Repair/drug effects , DNA Repair/genetics , Demethylation/drug effects , Fanconi Anemia Complementation Group Proteins/genetics , Fanconi Anemia Complementation Group Proteins/metabolism , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/metabolism , Humans , Hydrogen Peroxide/pharmacology , MAP Kinase Signaling System/drug effects , Methylation/drug effects , Mice , Phosphorylation , Protein Processing, Post-Translational , RNA, Small Interfering , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , RNA-Seq , Sumoylation/drug effects , Tandem Mass Spectrometry , X-linked Nuclear Protein/genetics , X-linked Nuclear Protein/metabolismABSTRACT
Coal seams were buried extremely shallowly in the trench slope area, which is prone to inducing surface cracks, seriously threatening the surface environment and mine safety production. The development of surface mining cracks varies at different locations in the trench slope area. In this research, we aimed to study the dynamic characteristics and laws of surface crack widths at different mining locations in the trench slope area and reveal the evolution mechanism of surface crack widths. Taking the 125,203 working face in Anshan Coal Mine in Shaanxi Province, China, as the geological prototype, we analyzed the full-cycle dynamic change law and planar distribution law of the surface crack widths in the trench slope area by combining the unmanned aerial vehicle remote sensing technology and field actual measurements and revealed the dynamic evolution mechanism of surface mining cracks in the trench area. The research results showed that the dynamic changes of surface crack widths varied at different locations of the slope. The surface crack width in the downslope area increased first and then stabilized with the advance of the working surface; the crack width at the bottom of the trench shows the dynamic change characteristic of increasing-decreasing-slightly increasing-stabilizing with the continuous advance of the working surface. The surface crack width in the upslope area showed the dynamic change of increasing-decreasing-stabilizing with the continuous advance of the working surface. Influenced by the surface morphology, the development mechanisms of surface mining cracks were different. The research results can provide practical guidance for selecting the best treatment time for surface cracks in different areas.
Subject(s)
Coal Mining , Coal Mining/methods , Coal/analysis , ChinaABSTRACT
N6-Methyladenosine (m6A) is the most abundant internal RNA modification catalyzed by host RNA methyltransferases. As obligate intracellular parasites, many viruses acquire m6A methylation in their RNAs. However, the biological functions of viral m6A methylation are poorly understood. Here, we found that viral m6A methylation serves as a molecular marker for host innate immunity to discriminate self from nonself RNA and that this novel biological function of viral m6A methylation is universally conserved in several families in nonsegmented negative-sense (NNS) RNA viruses. Using m6A methyltransferase (METTL3) knockout cells, we produced m6A-deficient virion RNAs from the representative members of the families Pneumoviridae, Paramyxoviridae, and Rhabdoviridae and found that these m6A-deficient viral RNAs triggered significantly higher levels of type I interferon compared to the m6A-sufficient viral RNAs, in a RIG-I-dependent manner. Reconstitution of the RIG-I pathway revealed that m6A-deficient virion RNA induced higher expression of RIG-I, bound to RIG-I more efficiently, enhanced RIG-I ubiquitination, and facilitated RIG-I conformational rearrangement and oligomerization. Furthermore, the m6A binding protein YTHDF2 is essential for suppression of the type I interferon signaling pathway, including by virion RNA. Collectively, our results suggest that several families in NNS RNA viruses acquire m6A in viral RNA as a common strategy to evade host innate immunity.IMPORTANCE The nonsegmented negative-sense (NNS) RNA viruses share many common replication and gene expression strategies. There are no vaccines or antiviral drugs for many of these viruses. We found that representative members of the families Pneumoviridae, Paramyxoviridae, and Rhabdoviridae among the NNS RNA viruses acquire m6A methylation in their genome and antigenome as a means to escape recognition by host innate immunity via a RIG-I-dependent signaling pathway. Viral RNA lacking m6A methylation induces a significantly higher type I interferon response than m6A-sufficient viral RNA. In addition to uncovering m6A methylation as a common mechanism for many NNS RNA viruses to evade host innate immunity, this study discovered a novel strategy to enhance type I interferon responses, which may have important applications in vaccine development, as robust innate immunity will likely promote the subsequent adaptive immunity.
Subject(s)
Adenosine/analogs & derivatives , Host Microbial Interactions/immunology , Interferon Type I/immunology , Negative-Sense RNA Viruses , RNA Virus Infections , RNA, Viral/genetics , A549 Cells , Adenosine/genetics , Gene Expression Regulation, Viral , Gene Knockout Techniques , Humans , Immunity, Innate , Methyltransferases/genetics , Negative-Sense RNA Viruses/genetics , Negative-Sense RNA Viruses/immunology , Negative-Sense RNA Viruses/pathogenicity , RNA Processing, Post-Transcriptional , RNA Virus Infections/immunology , RNA Virus Infections/virologyABSTRACT
Regulation of gene expression plays a fundamental role in cardiac stress-responses. Modification of coding transcripts by adenosine methylation (m6A) has recently emerged as a critical post-transcriptional mechanism underlying heart disease. Thousands of mammalian mRNAs are known to be m6A-modified, suggesting that remodeling of the m6A landscape may play an important role in cardiac pathophysiology. Here we found an increase in m6A content in human heart failure samples. We then adopted genome-wide analysis to define all m6A-regulated sites in human failing compared to non-failing hearts and identified targeted transcripts involved in histone modification as enriched in heart failure. Further, we compared all m6A sites regulated in human hearts with the ones occurring in isolated rat hypertrophic cardiomyocytes to define cardiomyocyte-specific m6A events conserved across species. Our results identified 38 shared transcripts targeted by m6A during stress conditions, and 11 events that are unique to unstressed cardiomyocytes. Of these, further evaluation of select mRNA and protein abundances demonstrates the potential impact of m6A on post-transcriptional regulation of gene expression in the heart.
Subject(s)
Adenosine/analogs & derivatives , Cardiomegaly/genetics , Myocardium/metabolism , Transcription, Genetic , Adenosine/metabolism , Animals , Animals, Newborn , Base Sequence , Biocatalysis , Heart Failure/genetics , Humans , Myocytes, Cardiac/pathology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Stress, Physiological/geneticsABSTRACT
BACKGROUND: Squamous cell carcinoma (SCC) is a disease with distinct management complexities as it displays a remarkably heterogeneous molecular subtype. However, the landscape of angiogenesis for SCC is not fully investigated. METHOD AND MATERIALS: The angiogenesis-related subtypes of SCC were established by using the ConsensusClusterPlus package based on angiogenesis-related genes and TCGA data. We analyzed the alteration of genes and miRNAs as well as pathways associated with angiogenesis subtypes. Next, the regulation network, the correlation with genomic characteristics, immune microenvironment, and clinical features of the angiogenesis subtypes were further investigated. Finally, the prognostic impact of the angiogenesis-related subtypes for SCC was also analyzed. RESULTS: A total of 1368 SCC samples were included in this study. Two angiogenesis subtypes were then identified based on the one hundred and sixty-three angiogenesis-related genes with subtype1 (angiogenesis subtype) of 951 SCC patients and subtype2 (non-angiogenesis subtype) of 417 SCC. GSEA revealed that angiogenesis and epithelial-mesenchymal transition, inflammatory response, and hypoxia were enriched in the angiogenesis subtype. Eight of the 15 immune checkpoints (ADORA2A, BTLA, CD276, CYBB, HAVCR2, SIGLEC7, SIGLEC9, and VTCN1) were significantly upregulated while C10orf54 were significantly downregulated in the angiogenesis subtype. The survival analysis revealed that the patients in the angiogenesis subtype have poorer survival outcomes than those in the non-angiogenesis subtype (P = 0.017 for disease-free interval and P = 0.00013 for overall survival). CONCLUSION: Our analysis revealed a novel angiogenesis subtype classification in SCC and provides new insights into a hallmark of SCC progression.
Subject(s)
Carcinoma, Squamous Cell , MicroRNAs , Antigens, CD , B7 Antigens , Carcinoma, Squamous Cell/genetics , Humans , Prognosis , Sialic Acid Binding Immunoglobulin-like Lectins , Survival Analysis , Tumor MicroenvironmentABSTRACT
The noncoding sequences in the UTR between the Gene Start signal (GS) and the initial codon AUG from 25 measles virus genomes were compared. The conserved sequences 5'-U(NNNNN)5-8-3' and 5'-(CNNNNNC)-3' were found in the genome and the UTR of phosphoprotein (P) and Fusion (F) mRNA. These sequences were named the U-Unit and the C-Unit, respectively. These two sequences are different in the P and F mRNAs. To investigate whether these conserved sequences can be used to regulate expression of foreign proteins in additional transcription units (ATU), a series of minigenomes with various 5' UTRs were generated. From reporter gene assays and quantitation of gene copies by qPCR, we found that the introduction of the C-unit into the 5'UTR of mRNA can down-regulate the expression of foreign proteins in additional transcription units in vivo.
Subject(s)
5' Untranslated Regions/genetics , Conserved Sequence/genetics , Down-Regulation/genetics , Gene Expression Regulation, Viral , Genome, Viral , Measles virus/genetics , Proteins/metabolism , Transcription, Genetic , Animals , Base Sequence , Cell Line , Codon/genetics , Cricetinae , Genes, Reporter , Nucleotides/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
This study was aimed to compare the mechanical characteristics under different physiological load conditions with three-dimensional finite element model of rigid fixation and elastic fixation in the lumbar. We observed the stress distribution characteristics of a sample of healthy male volunteer modeling under vertical, flexion and extension torque situation. The outcomes showed that there existed 4-6 times pressure on the connecting rod of rigid fixation compared with the elastic fixations under different loads, and the stress peak and area of force on elastic fixation were much higher than that of the rigid fixations. The elastic fixation has more biomechanical advantages than rigid fixation in promoting interbody lumbar fusion after surgery.
Subject(s)
Lumbar Vertebrae/surgery , Range of Motion, Articular , Spinal Fusion , Biomechanical Phenomena , Finite Element Analysis , Humans , Male , Models, Theoretical , PressureABSTRACT
Currently used directing groups for selective aliphatic ß-functionalization of carbonyl compounds show excellent reactivity and selectivity with an amide as a linker. Described herein is 2-piconimide, used for the first time with commercially available 2-picolinamide/2-picolic acid as precursors, to direct C-H arylation/alkenylation by palladium catalysis. The directing group is essential for promoting the sequnetial primary and secondary C(sp(3))-H arylation with different aryl iodides in one substrate. The directing group was easily removed under simple reaction conditions at room temperature.
ABSTRACT
Pseudouridine (Ψ) is an abundant RNA modification that is present in and affects the functions of diverse non-coding RNA species, including rRNA, tRNA and small nuclear RNA. Ψ also exists in mammalian mRNA and probably exhibits functional roles; however, functional investigations of mRNA Ψ modifications in mammals have been hampered by the lack of a quantitative method that detects Ψ at base precision. We have recently developed bisulfite-induced deletion sequencing (BID-seq), which provides the community with a quantitative method to map RNA Ψ distribution transcriptome-wide at single-base resolution. Here, we describe an optimized BID-seq protocol for mapping Ψ distribution across cellular mRNAs, which includes fast steps in both library preparation and data analysis. This protocol generates highly reproducible results by inducing high deletion ratios at Ψ modification within diverse sequence contexts, and meanwhile displayed almost zero background deletions at unmodified uridines. When used for transcriptome-wide Ψ profiling in mouse embryonic stem cells, the current protocol uncovered 8,407 Ψ sites from as little as 10 ng of polyA+ RNA input. This optimized BID-seq workflow takes 5 days to complete and includes four main sections: RNA preparation, library construction, next-generation sequencing (NGS) and data analysis. Library construction can be completed by researchers who have basic knowledge and skills in molecular biology and genetics. In addition to the experimental protocol, we provide BID-pipe ( https://github.com/y9c/pseudoU-BIDseq ), a user-friendly data analysis pipeline for Ψ site detection and modification stoichiometry quantification, requiring only basic bioinformatic and computational skills to uncover Ψ signatures from BID-seq data.
Subject(s)
Pseudouridine , Transcriptome , Animals , Mice , Pseudouridine/analysis , Pseudouridine/genetics , RNA, Messenger/genetics , Gene Expression Profiling/methods , RNA, Ribosomal/genetics , Mammals/geneticsABSTRACT
N6-methyladenosine (m6A) plays critical roles in regulating mRNA metabolism. However, comprehensive m6A methylomes in different plant tissues with single-base precision have yet to be reported. Here, we present transcriptome-wide m6A maps at single-base resolution in different tissues of rice and Arabidopsis using m6A-SAC-seq. Our analysis uncovers a total of 205,691 m6A sites distributed across 22,574 genes in rice, and 188,282 m6A sites across 19,984 genes in Arabidopsis. The evolutionarily conserved m6A sites in rice and Arabidopsis ortholog gene pairs are involved in controlling tissue development, photosynthesis and stress response. We observe an overall mRNA stabilization effect by 3' UTR m6A sites in certain plant tissues. Like in mammals, a positive correlation between the m6A level and the length of internal exons is also observed in plant mRNA, except for the last exon. Our data suggest an active m6A deposition process occurring near the stop codon in plant mRNA. In addition, the MTA-installed plant mRNA m6A sites correlate with both translation promotion and translation suppression, depicting a more complicated regulatory picture. Our results therefore provide in-depth resources for relating single-base resolution m6A sites with functions in plants and uncover a suppression-activation model controlling m6A biogenesis across species.