Chemical manipulation of m1A mediates its detection in human tRNA.

N 1-methyl adenosine (m1A) is a widespread RNA modification present in tRNA, rRNA, and mRNA. m1A modification sites in tRNAs are evolutionarily conserved and its formation on tRNA is catalyzed by methyltransferase TRMT61A and TRMT6 complex. m1A promotes translation initiation and elongation. Due to its positive charge under physiological conditions, m1A can notably modulate RNA structure. It also blocks Watson-Crick-Franklin base-pairing and causes mutation and truncation during reverse transcription. Several misincorporation-based high-throughput sequencing methods have been developed to sequence m1A. In this study, we introduce a reduction-based m1A sequencing (red-m1A-seq). We report that NaBH4 reduction of m1A can improve the mutation and readthrough rates using commercially available RT enzymes to give a better positive signature, while alkaline-catalyzed Dimroth rearrangement can efficiently convert m1A to m6A to provide good controls, allowing the detection of m1A with higher sensitivity and accuracy. We applied red-m1A-seq to sequence human small RNA, and we not only detected all the previously reported tRNA m1A sites, but also new m1A sites in mt-tRNAAsn-GTT and 5.8S rRNA.

Sequencing of N6-methyl-deoxyadenosine at single-base resolution across the mammalian genome.

Although DNA N6-methyl-deoxyadenosine (6mA) is abundant in bacteria and protists, its presence and function in mammalian genomes have been less clear. We present Direct-Read 6mA sequencing (DR-6mA-seq), an antibody-independent method, to measure 6mA at base resolution. DR-6mA-seq employs a unique mutation-based strategy to reveal 6mA sites as misincorporation signatures without any chemical or enzymatic modulation of 6mA. We validated DR-6mA-seq through the successful mapping of the well-characterized G(6mA)TC motif in the E. coli DNA. As expected, when applying DR-6mA-seq to mammalian systems, we found that genomic DNA (gDNA) 6mA abundance is generally low in most mammalian tissues and cells; however, we did observe distinct gDNA 6mA sites in mouse testis and glioblastoma cells. DR-6mA-seq provides an enabling tool to detect 6mA at single-base resolution for a comprehensive understanding of DNA 6mA in eukaryotes.

BID-seq for transcriptome-wide quantitative sequencing of mRNA pseudouridine at base resolution.

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.

Regulation of forkhead box O transcription factor by insulin signaling pathway controls the reproductive diapause of the lady beetle, Coccinella septempunctata.

In biological control programs, knowledge about diapause regulation in natural enemy insects provides important insight for improving long-term storage, transportation, and field adoption of these biological control agents. As a natural predator of agricultural pests, the lady beetle Coccinella septempunctata has been commercially mass-cultured and widely employed in pest management. In some insects, insulin signaling, in conjunction with the downstream transcription factor Forkhead box O (FoxO), are master regulators of multiple physiological processes involved in diapause, but it is unclear whether insulin signaling and FoxO affect the diapause of C. septempunctata. In this study, we use a combination of approaches to demonstrate that insulin signaling and FoxO mediate the diapause response in C. septempunctata. In diapausing beetles, application of exogenous insulin and knocking down expression of CsFoxo with RNA interference (RNAi) both rescued beetles from developmental arrest. In non-diapausing beetles, knocking down expression of the insulin receptor (CsInR) with RNA interference (RNAi) arrested ovarian development and decreased juvenile hormone (JH) content to levels comparable to the diapause state. Taken together, these results suggest that a shutdown of insulin signaling prompts the activation of the downstream FoxO gene, leading to the diapause phenotype.

Male vitellogenin regulates gametogenesis through a testis-enriched big protein in Chrysopa pallens.

In insects, vitellogenin (Vg) is generally viewed as a female-specific protein. Its primary function is to supply nutrition to developing embryos. Here, we reported Vg from the male adults of a natural predator, Chrysopa pallens. The male Vg was depleted by RNAi. Mating with Vg-deficient male downregulated female Vg expression, suppressed ovarian development and decreased reproductive output. Whole-organism transcriptome analysis after male Vg knockdown showed no differential expression of the known spermatogenesis-related regulators and seminal fluid protein genes, but a sharp downregulation of an unknown gene, which encodes a testis-enriched big protein (Vcsoo). Separate knockdown of male Vg and Vcsoo disturbed the assembly of spermatid cytoplasmic organelles in males and suppressed the expansion of ovary germarium in mated females. These results demonstrated that C. pallens male Vg signals through the downstream Vcsoo and regulates male and female reproduction.

Base-Resolution Sequencing Methods for Whole-Transcriptome Quantification of mRNA Modifications.

ConspectusRNA molecules are not merely a combination of four bases of A, C, G, and U. Chemical modifications occur in almost all RNA species and play diverse roles in gene expression regulation. The abundant cellular RNAs, such as ribosomal RNA (rRNA) and transfer RNA (tRNA), are known to have the highest density of RNA modifications, which exert critical functions in rRNA and tRNA biogenesis, stability, and subsequent translation. In recent years, modifications on low-abundance RNA species in mammalian cells, such as messenger RNA (mRNA), regulatory noncoding RNA (ncRNA), and chromatin-associated RNA (caRNA), have been shown to contain multiple different chemical modifications with functional significance.As the most abundant mRNA modification in mammals, N6-methyladenosine (m6A) affects nearly every stage of mRNA processing and metabolism, with the antibody-based m6A-MeRIP-seq (methylated RNA immunoprecipitation sequencing) followed by high-throughput sequencing widely employed in mapping m6A distribution transcriptome-wide in diverse biological systems. In addition to m6A, other chemical modifications such as pseudouridine (Ψ), 2'-O-methylation (Nm), 5-methylcytidine (m5C), internal N7-methylguanosine (m7G), N1-methyladenosine (m1A), N4-acetylcytidine (ac4C), etc. also exist in polyA-tailed RNA in mammalian cells, requiring effective mapping approaches for whole-transcriptome profiling of these non-m6A mRNA modifications. Like m6A, the antibody-based enrichment followed by sequencing has been the primary method to study distributions of these modifications. Methods to more quantitatively map these modifications would dramatically improve our understanding of distributions and modification density of these chemical marks on RNA, thereby bettering informing functional implications. In this Account, aimed at both single-base resolution and modification fraction quantification, we summarize our recent advances in developing a series of chemistry- or biochemistry-based methods to quantitatively map RNA modifications, including m6A, Ψ, m5C, m1A, 2'-O-methylation (Nm), and internal m7G, in mammalian mRNA at base resolution. These new methods, including m6A-SAC-seq, eTAM-seq, BID-seq, UBS-seq, DAMM-seq, m1A-quant-seq, Nm-Mut-seq, and m7G-quant-seq, promise to conduct base-resolution mapping of most major mRNA modifications with low RNA input and uncover dynamic changes in modification stoichiometry during biological and physiological processes, facilitating future investigations on these RNA modifications in regulating cellular gene expression and as potential biomarkers for clinical diagnosis and prognosis. These quantitative sequencing methods allow the mapping of most mRNA modifications with limited input sample requirements. The same modifications on diverse RNA species, such as caRNA, ncRNA, nuclear nascent RNA, mitochondrial RNA, cell-free RNA (cfRNA), etc., could be sequenced using the same methods.

RNA Interference in the Egg Parasitoid, Trichogramma dendrolimi Matsumura.

The egg parasitoids, Trichogramma spp, are recognized as efficient biological control agents against various lepidopteran pests in agriculture and forests. The immature stages of Trichogramma offspring develop within the host egg, exhibiting remarkable diminutiveness (approximately 0.5 mm in adult length). RNA-interference (RNAi) methodology has emerged as a crucial tool for elucidating gene functions in numerous organisms. However, manipulating RNAi in certain small parasitoid species, such as Trichogramma, has generally posed significant challenges. In this study, we present an efficient RNAi method in Trichogramma denrolimi. The outlined procedure encompasses the acquisition and isolation of individual T. dendrolimi specimens from host eggs, the design and synthesis of double-stranded RNA (dsRNA), the in vitro transplantation and cultivation of T. dendrolimi pupae, the micro-injection of dsRNA, and the subsequent assessment of target gene knockdown through RT-qPCR analysis. This study furnishes a comprehensive, visually detailed procedure for conducting RNAi experiments in T. dendrolimi, thereby enabling researchers to investigate the gene regulation in this species. Furthermore, this methodology is adaptable for RNAi studies or micro-injections in other Trichogramma species with minor adjustments, rendering it a valuable reference for conducting RNAi experiments in other endoparasitic species.

Quantitative base-resolution sequencing technology for mapping pseudouridines in mammalian mRNA.

Posttranscriptional RNA modifications occur in almost all types of RNA in all life forms. As an abundant RNA modification in mammals, pseudouridine (Ψ) regulates diverse biological functions of different RNA species such as ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), etc. However, the functional investigation of mRNA pseudouridine (Ψ) has been hampered by the lack of a quantitative method that can efficiently map Ψ transcriptome-wide. We developed bisulfite-induced deletion sequencing (BID-seq), with the optimized bisulfite-based chemical reaction to convert pseudouridine selectively and completely into Ψ-BS adduct without cytosine deamination. The Ψ-BS adduct can be further read out as deletion signatures during reverse transcription. The deletion ratios induced by Ψ sites were used for estimating the modification stoichiometry at each modified site. BID-seq starts with 10-20 ng polyA+ RNA and detects thousands of mRNA Ψ sites with stoichiometry information in cell lines and tissues. We uncovered consensus motifs for Ψ in mammalian mRNA and assigned specific 'writer' proteins to individual Ψ deposition. BID-seq also confirmed the presence of Ψ within stop codons of mammalian mRNA. BID-seq set the stage for future investigations of Ψ functions in diverse biological processes.

Base-resolution quantitative DAMM-seq for mapping RNA methylations in tRNA and mitochondrial polycistronic RNA.

The human AlkB family proteins, such as FTO and ALKBH5, are known to mediate RNA m6A demethylation. However, although ALKBH7 localizes in mitochondria and affects metabolism, the detailed biological function and mechanism have remained unknown for years. We developed Demethylation-Assisted Multiple Methylation sequencing (DAMM-seq) to simultaneously detect N1-methyladenosine (m1A), N3-methylcytidine (m3C), N1-methylguanosine (m1G) and N2,N2-dimethylguanosine (m22G) methylations in both steady-state RNA and nascent RNA, and discovered that human ALKBH7 demethylates m22G and m1A within mt-Ile and mt-Leu1 pre-tRNA regions, respectively, in mitochondrial polycistronic RNA. DAMM-seq quantitatively and sensitively monitors the methylation stoichiometry change at pre-tRNA junctions within nascent mt-RNA, revealing the target region where ALKBH7 regulates RNA processing and local structural switch of polycistronic mt-RNAs. A new RNA demethylase in human cells was characterized through the base-resolution quantification of multiple RNA methylations in nascent mt-RNA, resolving the long-standing question about the functional substrate of ALKBH7.

The regulation of antiviral innate immunity through non-m6A RNA modifications.

The post-transcriptional RNA modifications impact the dynamic regulation of gene expression in diverse biological and physiological processes. Host RNA modifications play an indispensable role in regulating innate immune responses against virus infection in mammals. Meanwhile, the viral RNAs can be deposited with RNA modifications to interfere with the host immune responses. The N6-methyladenosine (m6A) has boosted the recent emergence of RNA epigenetics, due to its high abundance and a transcriptome-wide widespread distribution in mammalian cells, proven to impact antiviral innate immunity. However, the other types of RNA modifications are also involved in regulating antiviral responses, and the functional roles of these non-m6A RNA modifications have not been comprehensively summarized. In this Review, we conclude the regulatory roles of 2'-O-methylation (Nm), 5-methylcytidine (m5C), adenosine-inosine editing (A-to-I editing), pseudouridine (Ψ), N1-methyladenosine (m1A), N7-methylguanosine (m7G), N6,2'-O-dimethyladenosine (m6Am), and N4-acetylcytidine (ac4C) in antiviral innate immunity. We provide a systematic introduction to the biogenesis and functions of these non-m6A RNA modifications in viral RNA, host RNA, and during virus-host interactions, emphasizing the biological functions of RNA modification regulators in antiviral responses. Furthermore, we discussed the recent research progress in the development of antiviral drugs through non-m6A RNA modifications. Collectively, this Review conveys knowledge and inspiration to researchers in multiple disciplines, highlighting the challenges and future directions in RNA epitranscriptome, immunology, and virology.

The identification and expression pattern of the sex determination genes and their sex-specific variants in the egg parasitoid Trichogramma dendrolimi Matsumura (Hymenoptera: Trichogrammatidae).

Introduction: Trichogramma wasps are egg parasitoids of agricultural lepidopteran pests. The sex of Trichogramma is determined by its ploidy as well as certain sex ratio distorters, such as the endosymbiotic bacteria Wolbachia spp. and the paternal sex ratio (PSR) chromosome. The sex determination systems of hymenopterans, such as Trichogramma spp., involve cascades of the genes transformer (tra), transformer-2 (tra2), and doublesex (dsx) and are associated with sex-specific tra and dsx splicing. First, these genes and their sex-specific variants must be identified to elucidate the interactions between the sex ratio disorders and the sex determination mechanism of Trichogramma. Methods: Here, we characterized the sex determination genes tra, tra2, and dsx in Trichogramma dendrolimi. Sex-specific tra and dsx variants were detected in cDNA samples obtained from both male and female Trichogramma wasps. They were observed in the early embryos (1-10 h), late embryos (12-20 h), larvae (32 h and 48 h), pre-pupae (96 h), and pupae (144 h, 168 h, 192 h, and 216 h) of both male and female T. dendrolimi offspring. Results: We detected female-specific tra variants throughout the entire early female offspring stage. The male-specific variant began to express at 9-10 h as the egg was not fertilized. However, we did not find any maternally derived, female-specific tra variant in the early male embryo. This observation suggests that the female-specific tra variant expressed in the female embryo at 1-9 h may not have originated from the maternal female wasp. Discussion: The present study might be the first to identify the sex determination genes and sex-specific gene splicing in Trichogramma wasps. The findings of this study lay the foundation for investigating the sex determination mechanisms of Trichogramma and other wasps. They also facilitate sex identification in immature T. dendrolimi and the application of this important egg parasitoid in biological insect pest control programs.

Posterior concentration of Wolbachia during the early embryogenesis of the host dynamically shapes the tissue tropism of Wolbachia in host Trichogramma wasps.

Introduction: The bacterial endosymbiont, Wolbachia spp. induce thelytokous parthenogenesis in certain parasitoid wasps, such as the egg parasitoid wasps Trichogramma spp. To complete the cycle of vertical transmission, Wolbachia displays efficient transovarial transmission by targeting the reproductive tissues and often exhibits strong tissue-specific tropism in their host. Method: The present study aimed to describe the basic Wolbachia distribution patterns that occur during the development of Wolbachia-infected, thelytokous Trichogramma dendrolimi, and T. pretiosum. We used fluorescence in situ hybridization (FISH) to investigate Wolbachia signal dynamics during early embryogenesis (from 30 to 120 min). Wolbachia titers and distributions from the embryo to adult stages of Trichogramma after early embryogenesis were detected by absolute quantitative polymerase chain reaction (AQ-PCR) and FISH. The symmetry ratios (SR) of the Wolbachia signals were calculated using the SR odds ratios in the anterior and posterior parts of the host. The SR was determined to describe Wolbachia tropism during early embryogenesis and various developmental stages of Trichogramma. Results: Wolbachia was concentrated in the posterior part of the embryo during early embryogenesis and the various developmental stages of both T. dendrolimi and T. pretiosum. Wolbachia density increased with the number of nuclei and the initial mitotic division frequency during early embryogenesis. The total Wolbachia titer increased with postembryogenesis development in both T. dendrolimi and T. pretiosum. However, the Wolbachia densities relative to body size were significantly lower at the adult and pupal stages than they were at the embryonic stage. Discussion: The present work revealed that posterior Wolbachia concentration during early host embryogenesis determined Wolbachia localization in adult wasps. By this mechanism, Wolbachia exhibits efficient vertical transmission across generations by depositing only female Wolbachia-infected offspring. The results of this study describe the dynamics of Wolbachia during the development of their Trichogramma host. The findings of this investigation helped clarify Wolbachia tropism in Trichogramma wasps.

Exon architecture controls mRNA m6A suppression and gene expression.

N6-methyladenosine (m6A) is the most abundant messenger RNA (mRNA) modification and plays crucial roles in diverse physiological processes. Using a massively parallel assay for m6A (MPm6A), we discover that m6A specificity is globally regulated by suppressors that prevent m6A deposition in unmethylated transcriptome regions. We identify exon junction complexes (EJCs) as m6A suppressors that protect exon junction-proximal RNA within coding sequences from methylation and regulate mRNA stability through m6A suppression. EJC suppression of m6A underlies multiple global characteristics of mRNA m6A specificity, with the local range of EJC protection sufficient to suppress m6A deposition in average-length internal exons but not in long internal and terminal exons. EJC-suppressed methylation sites colocalize with EJC-suppressed splice sites, which suggests that exon architecture broadly determines local mRNA accessibility to regulatory complexes.

14-3-3ζ inhibits maladaptive repair in renal tubules by regulating YAP and reduces renal interstitial fibrosis.

Acute kidney injury (AKI) refers to a group of common clinical syndromes characterized by acute renal dysfunction, which may lead to chronic kidney disease (CKD), and this process is called the AKI-CKD transition. The transcriptional coactivator YAP can promote the AKI-CKD transition by regulating the expression of profibrotic factors, and 14-3-3 protein zeta (14-3-3ζ), an important regulatory protein of YAP, may prevent the AKI-CKD transition. We established an AKI-CKD model in mice by unilateral renal ischemia-reperfusion injury and overexpressed 14-3-3ζ in mice using a fluid dynamics-based gene transfection technique. We also overexpressed and knocked down 14-3-3ζ in vitro. In AKI-CKD model mice, 14-3-3ζ expression was significantly increased at the AKI stage. During the development of chronic disease, the expression of 14-3-3ζ tended to decrease, whereas active YAP was consistently overexpressed. In vitro, we found that 14-3-3ζ can combine with YAP, promote the phosphorylation of YAP, inhibit YAP nuclear translocation, and reduce the expression of fibrosis-related proteins. In an in vivo intervention experiment, we found that the overexpression of 14-3-3ζ slowed the process of renal fibrosis in a mouse model of AKI-CKD. These findings suggest that 14-3-3ζ can affect the expression of fibrosis-related proteins by regulating YAP, inhibit the maladaptive repair of renal tubular epithelial cells, and prevent the AKI-CKD transition.

The green lacewing Chrysopa formosa as a potential biocontrol agent for managing Spodoptera frugiperda and Spodoptera litura.

Understanding predator-prey interactions is essential for successful pest management by using predators, especially for the suppression of novel invasive pest. The green lacewing Chrysopa formosa is a promising polyphagous predator that is widely used in the biocontrol of various pests in China, but information on the control efficiency of this predator against the seriously invasive pest Spodoptera frugiperda and native Spodoptera litura is limited. Here we evaluated the predation efficiency of C. formosa adults on eggs and first- to third-instar larvae of S. frugiperda and S. litura through functional response experiments and determined the consumption capacity and prey preference of this chrysopid. Adults of C. formosa had a high consumption of eggs and earlier instar larvae of both prey species, and displayed a type II functional response on all prey stages. Attack rates of the chrysopid on different prey stages were statistically similar, but the handling time increased notably as the prey developed. The highest predation efficiency and shortest-handling time were observed for C. formosa feeding on Spodoptera eggs, followed by the first-instar larvae. C. formosa exhibited a significant preference for S. litura over S. frugiperda in a two-prey system. In addition, we summarized the functional response and predation efficiency of several chrysopids against noctuid pests and made a comparison with the results obtained from C. formosa. These results indicate that C. formosa has potential as an agent for biological control of noctuid pests, particularly for the newly invasive pest S. frugiperda in China.

BID-seq: The Quantitative and Base-Resolution Sequencing Method for RNA Pseudouridine.

Quantitative and base-resolution sequencing methods are critical to investigations of the biological functions of diverse RNA modifications. These methods may also be employed for clinical studies and clinical applications in the future. In this In Focus article, we introduce and discuss the development of Bisulfite-Induced Deletion sequencing (BID-seq) for quantitatively detecting mRNA pseudouridine (Ψ) modifications at base resolution.

Wolbachia-infected Trichogramma dendrolimi is outcompeted by its uninfected counterpart in superparasitism but does not have developmental delay.

BACKGROUND: Wolbachia infection increases the superparasitism frequency of Trichogramma females and provides an opportunity for horizontal intraspecific transmission. However, superparasitism may lead to interstrain competition between Wolbachia-infected Trichogramma offspring and their uninfected counterparts. This study investigated the outcome of interstrain intrinsic competition between Wolbachia-infected thelytokous strain (W) and uninfected bisexual strain (B) of Trichogramma dendrolimi. To determine the developmental rate of both strains, the sizes of immature stages of T. dendrolimi offspring at different times after parasitisation were measured in single parasitism and superparasitism conditions. RESULTS: The results reflect increased superparasitism by Wolbachia-infected females compared with uninfected females. Trichogramma females did not discriminate between host eggs previously parasitised by either B or W females. When the first oviposition was performed by B females, the B offspring outcompeted W offspring deposited later. Although when W offspring was deposited 8 h earlier than the B offspring, it gained no advantage over B offspring. Regardless of parasitism conditions, differences in the development rate between W and B offspring were not significant. CONCLUSION: The results reconfirmed that W females presented a higher tendency for superparasitism than B females, and showed that B offspring outcompeted W offspring even when the latter was deposited 8 h earlier. The inferiority of Wolbachia-infected Trichogramma compared with their uninfected counterparts is not due to the developmental delay. This study provides insights into the effects of intrinsic competition on the control efficacy of Wolbachia-infected Trichogramma against pests in biological control programs. © 2022 Society of Chemical Industry.

Quantitative sequencing using BID-seq uncovers abundant pseudouridines in mammalian mRNA at base resolution.

Functional characterization of pseudouridine (Ψ) in mammalian mRNA has been hampered by the lack of a quantitative method that maps Ψ in the whole transcriptome. We report bisulfite-induced deletion sequencing (BID-seq), which uses a bisulfite-mediated reaction to convert pseudouridine stoichiometrically into deletion upon reverse transcription without cytosine deamination. BID-seq enables detection of abundant Ψ sites with stoichiometry information in several human cell lines and 12 different mouse tissues using 10-20 ng input RNA. We uncover consensus sequences for Ψ in mammalian mRNA and assign different 'writer' proteins to individual Ψ deposition. Our results reveal a transcript stabilization role of Ψ sites installed by TRUB1 in human cancer cells. We also detect the presence of Ψ within stop codons of mammalian mRNA and confirm the role of Ψ in promoting stop codon readthrough in vivo. BID-seq will enable future investigations of the roles of Ψ in diverse biological processes.

m7G-quant-seq: Quantitative Detection of RNA Internal N7-Methylguanosine.

Methods for the precise detection and quantification of RNA modifications are critical to uncover functional roles of diverse RNA modifications. The internal m7G modification in mammalian cytoplasmic tRNAs is known to affect tRNA function and impact embryonic stem cell self-renewal, tumorigenesis, cancer progression, and other cellular processes. Here, we introduce m7G-quant-seq, a quantitative method that accurately detects internal m7G sites in human cytoplasmic tRNAs at single-base resolution. The efficient chemical reduction and mild depurination can almost completely convert internal m7G sites into RNA abasic sites (AP sites). We demonstrate that RNA abasic sites induce a mixed variation pattern during reverse transcription, including G → A or C or T mutations as well as deletions. We calculated the total variation ratio to quantify the m7G modification fraction at each methylated site. The calibration curves of all relevant motif contexts allow us to more quantitatively determine the m7G methylation level. We detected internal m7G sites in 22 human cytoplasmic tRNAs from HeLa and HEK293T cells and successfully estimated the corresponding m7G methylation stoichiometry. m7G-quant-seq could be applied to monitor the tRNA m7G methylation level change in diverse biological processes.