The role of N6‐methyladenosine modification on diapause in silkworm (Bombyx mori) strains that exhibit different voltinism

N6‐methyladenosine (m⁶A) plays a key role in regulating gene expression in myriad organisms. Diapause is an important plastic phenotype that allows insects to survive under specific environmental conditions. However, the diapause molecular mechanism remains unknown. In this study, we analyzed the phylogenetics of genes related to the m⁶A modification complex in the silkworm (Bombyx mori) based on identified sequences from other organisms. We detected the expression of these genes during different developmental phases from four strains with different voltinism. We also determined total m⁶A content in cells treated with different diapause hormone concentrations or eggs exposed to hydrochloric acid. Our data revealed that m⁶A‐modification‐related gene expression and m⁶A content were greater in diapause‐destinated compared to nondiapause‐destined strains. Our findings suggest that m⁶A modification may provide significant epigenetic regulation of diapause‐related genes in the silkworm.     

The yfiC gene of E. coli encodes an adenine-N6 methyltransferase that specifically modifies A37 of tRNA1Val(cmo5UAC)

Transfer RNA is highly modified. Nucleotide 37 of the anticodon loop is represented by various modified nucleotides. In Escherichia coli, the valine-specific tRNA (cmo⁵UAC) contains a unique modification, N⁶-methyladenosine, at position 37; however, the enzyme responsible for this modification is unknown. Here we demonstrate that the yfiC gene of E. coli encodes an enzyme responsible for the methylation of A37 in tRNA₁^Val. Inactivation of yfiC gene abolishes m⁶A formation in tRNA₁^Val, while expression of the yfiC gene from a plasmid restores the modification. Additionally, unmodified tRNA₁^Val can be methylated by recombinant YfiC protein in vitro. Although the methylation of m⁶A in tRNA₁^Val by YfiC has little influence on the cell growth under standard conditions, the yfiC gene confers a growth advantage under conditions of osmotic and oxidative stress.     

N~6 -methyl-adenosine (m ~6A) in RNA: An Old Modification with A Novel Epigenetic Function

N6 -methyl-adenosine (m6A) is one of the most common and abundant modifications on RNA molecules present in eukaryotes. However, the biological significance of m6A methylation remains largely unknown. Several independent lines of evidence suggest that the dynamic regulation of m6A may have a profound impact on gene expression regulation. The m6A modification is catalyzed by an unidentified methyltransferase complex containing at least one subunit methyltransferase like 3 (METTL3). m6A modification on messenger RNAs (mRNAs) mainly occurs in the exonic regions and 3’-untranslated region (3’-UTR) as revealed by high-throughput m6A-seq. One significant advance in m6A research is the recent discovery of the first two m6A RNA demethylases fat mass and obesity-associated (FTO) gene and ALKBH5, which catalyze m6A demethylation in an a-ketoglutarate (a-KG)-and Fe2+-dependent manner. Recent studies in model organisms demonstrate that METTL3, FTO and ALKBH5 play important roles in many biological processes, ranging from development and metabolism to fertility. Moreover, perturbation of activities of these enzymes leads to the disturbed expression of thousands of genes at the cellular level, implicating a regulatory role of m6A in RNA metabolism. Given the vital roles of DNA and histone methylations in epigenetic regulation of basic life processes in mammals, the dynamic and reversible chemical m6A modification on RNA may also serve as a novel epigenetic marker of profound biological significances.     

Low METTL3 level in midgut of the Bombyx mori inhibit the proliferation of nucleopolyhedrovirus

N6-methyladeosine (m⁶A) plays an important role in virus infection and replication. Bombyx mori nuclear polyhedrosis is caused by Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Expression levels of m⁶A-modification-related genes after the infection of BmNPV were detected at first. Then, expression levels of BmNPV nucleocapsid protein gene VP39 and envelope fusion protein gene GP64 after knockdown of METTL3in vitro were quantified to identify the effect of m⁶A modification on BmNPV. BmNPV firstly infects the larval midgut in case of oral infection. Subsequently, to clarify the relationship between m⁶A modification and resistance of the silkworm to BmNPV, we detected the expression levels of m⁶A-modification-related genes invivo before and after infection of BmNPV. The results indicated that low METTL3 level hindered the proliferation of BmNPV to some extent, and silkworm strain with low METTL3 level showed stronger resistance against BmNPV. This study will accumulate new experimental data for elucidating the resistance mechanism of silkworm against BmNPV.     

RNA N6-methyladenosine modification suppresses replication of rice black streaked dwarf virus and is associated with virus persistence in its insect vector

N⁶ methylation of adenosine (m⁶A) was recently discovered to play a role in regulating the life cycle of various viruses by modifying viral and host RNAs. However, different studies on m⁶A effects on the same or different viruses have revealed contradictory roles for m⁶A in the viral life cycle. In this study, we sought to define the role of m⁶A on infection by rice black streaked dwarf virus (RBSDV), a double-stranded RNA virus, of its vector small brown planthopper (SBPH). Infection by RBSDV decreased the level of m⁶A in midgut cells of SBPHs. We then cloned two genes (LsMETTL3 and LsMETTL14) that encode m⁶A RNA methyltransferase in SBPHs. After interference with expression of the two genes, the titre of RBSDV in the midgut cells of SBPHs increased significantly, suggesting that m⁶A levels were negatively correlated with virus replication. More importantly, our results revealed that m⁶A modification might be the epigenetic mechanism that regulates RBSDV replication in its insect vector and maintains a certain virus threshold required for persistent transmission.     

Patterns of cytosine methylation in an elite rice hybrid and its parental lines, detected by a methylation-sensitive amplification polymorphism technique

DNA methylation is known to play an important role in the regulation of gene expression in eukaryotes. In this study, we assessed the extent and pattern of cytosine methylation in the rice genome, using the technique of methylation-sensitive amplified polymorphism (MSAP), which is a modification of the amplified fragment length polymorphism (AFLP) method that makes use of the differential sensitivity of a pair of isoschizomers to cytosine methylation. The tissues assayed included seedlings and flag leaves of an elite rice hybrid, Shanyou 63, and the parental lines Zhenshan 97 and Minghui 63. In all, 1076 fragments, each representing a recognition site cleaved by either or both of the isoschizomers, were amplified using 16 pairs of selective primers. A total of 195 sites were found to be methylated at cytosines in one or both parents, and the two parents showed approximately the same overall degree of methylation (16.3%), as revealed by the incidence of differential digestion by the isoschizomers. Four classes of patterns were identified in a comparative assay of cytosine methylation in the parents and hybrid; increased methylation was detected in the hybrid compared to the parents at some of the recognition sites, while decreased methylation in the hybrid was detected at other sites. A small proportion of the sites was found to be differentially methylated in seedlings and flag leaves; DNA from young seedlings was methylated to a greater extent than that from flag leaves. Almost all of the methylation patterns detected by MSAP could be confirmed by Southern analysis using the isolated amplified fragments as probes. The results clearly demonstrate that the MSAP technique is highly efficient for large-scale detection of cytosine methylation in the rice genome. We believe that the technique can be adapted for use in other plant species. Received: 23 October 1998 / Accepted: 11 January 1999     

Host plant volatiles responsible for the invasion of Stenotus rubrovittatus (Heteroptera: Miridae) into paddy fields

We investigated the attractiveness of synthetic volatile blends or individual volatiles of flowering rice panicles or flowering Scirpus juncoides spikelets to the sorghum plant bug Stenotus rubrovittatus (Matsumura). None of the individual chemicals tested attracted either sex of the bug. Synthetic volatile blends of flowering rice panicles composed of geranyl acetone, β‐caryophyllene, n‐decanal, methyl salicylate, β‐elemene and n‐tridecene attracted females. The synthetic blend of volatiles was just as attractive as natural flowering rice panicles to females. Other synthetic blends did not attract the bug. We sampled headspace volatiles from flowering S. juncoides spikelets with solid‐phase microextraction and analysed them using gas chromatography–mass spectrometry. The main volatile emitted from S. juncoides was β‐caryophyllene, one of the major volatile components of flowering rice panicles. β‐Elemene was a common volatile found in flowering rice panicles and flowering S. juncoides spikelets. Therefore, we investigated the attractiveness of synthetic blends of flowering rice panicles and S. juncoides spikelets composed of β‐caryophyllene and β‐elemene. The synthetic blend of flowering S. juncoides spikelets significantly attracted males but not females. The synthetic blend of flowering rice panicles composed of β‐caryophyllene and β‐elemene did not attract either sex. These results suggest that β‐caryophyllene and β‐elemene are common active compounds responsible for attractiveness of flowering rice panicles and S. juncoides spikelets although some of the other volatile components act synergistically with these two compounds in natural plant odours.     

A microcolinearity study at the earliness per se gene Eps-A m 1 region reveals an ancient duplication that preceded the wheat–rice divergence

Wheat flowering is controlled by numerous genes, which respond to environmental signals such as photoperiod and vernalization. Earliness per se (Eps) genes control flowering time independently of these environmental cues and are responsible for the fine tuning of flowering time. We recently mapped the Eps-A ^m 1 gene on the end of Triticum monococcum chromosome arm 1A^mL. As a part of our efforts to clone Eps-A ^m 1 we developed PCR markers flanking this gene within a 2.7 cM interval. We screened more than one thousand gametes with these markers and identified 27 lines with recombination between them. Recombinant lines were used to generate a high-density map and to investigate the microcolinearity between wheat and rice in this region. We mapped ten genes from a 149 kb region located at the distal part of rice chromosome 5 (cdo393 – Ndk3) on a 3.7 cM region on wheat chromosome one. This region is part of an ancient duplication between rice chromosomes 5 and 1. Genes present in both rice chromosomes were less similar to each other than to the closest wheat orthologues, suggesting that this duplication preceded the divergence between wheat and rice. This hypothesis was supported by the presence of 18 loci duplicated both in rice chromosomes 5 and 1 and in the colinear wheat chromosomes from homoeologous groups 1 and 3. Independent gene deletions in wheat and rice lineages explain the alternations of colinearity between rice chromosome 5 and wheat chromosomes 1 and 3. Colinearity between the end of rice chromosome 5 and wheat chromosome 1 was also interrupted by a small inversion, and several non-colinear genes. These results suggest that the distal region of the long arm of wheat chromosome 1 was involved in numerous changes that differentiated wheat and rice genomes. This comparative study provided sufficient markers to saturate the Eps-A ^m 1 gene region and to precisely map this gene within a 0.9 cM interval flanked by the VatpC and Smp loci. Sequences obtained in this study: DQ196178, DQ196179, DQ196180, DQ196181, DQ196182, DQ196183, DQ196184, DQ196185, DQ196186, DQ196187, DQ196488, DQ198537, DQ308530, DQ308531, DQ308532, DQ308533, DQ308534, DQ308535, DQ308536, DQ308537, DQ308538, DQ308539, DQ308540     

Novel insights into m6A modification of coding and non-coding RNAs in tumor biology: From molecular mechanisms to therapeutic significance

Accumulating evidence has revealed that m⁶A modification, the predominant RNA modification in eukaryotes, adds a novel layer of regulation to the gene expression. Dynamic and reversible m⁶A modification implements sophisticated and crucial functions in RNA metabolism, including generation, splicing, stability, and translation in messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs). Furthermore, m⁶A modification plays a determining role in producing various m⁶A-labeling RNA outcomes, thereby affecting several functional processes, including tumorigenesis and progression. Herein, we highlighted current advances in m⁶A modification and the regulatory mechanisms underlying mRNAs and ncRNAs in distinct cancer stages. Meanwhile, we also focused on the therapeutic significance of m⁶A regulators in clinical cancer treatment.     

METTL3-mediated mRNA N6-methyladenosine is required for oocyte and follicle development in mice

Proper follicle development is very important for the production of mature oocytes, which is essential for the maintenance of female fertility. This complex biological process requires precise gene regulation. The most abundant modification of mRNA, N⁶-methyladenosine (m⁶A), is involved in many RNA metabolism processes, including RNA splicing, translation, stability, and degradation. Here, we report that m⁶A plays essential roles during oocyte and follicle development. Oocyte-specific inactivation of the key m⁶A methyltransferase Mettl3 with Gdf9-Cre caused DNA damage accumulation in oocytes, defective follicle development, and abnormal ovulation. Mechanistically, combined RNA-seq and m⁶A methylated RNA immunoprecipitation sequencing (MeRIP-seq) data from oocytes revealed, that we found METTL3 targets Itsn2 for m⁶A modification and then enhances its stability to influence the oocytes meiosis. Taken together, our findings highlight the crucial roles of mRNA m⁶A modification in follicle development and coordination of RNA stabilization during oocyte growth.Subject terms: Oogenesis, Infertility