ALKBH1-Mediated tRNA Demethylation Regulates Translation.

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

The Bioinformatical Identification of Potential Biomarkers in Heart Failure Diagnosis and Treatment.

Background: Heart failure (HF) is defined as the inability of the heart's systolic and diastolic function to properly discharge blood flow from the veins to the heart. The goal of our research is to look into the possible mechanism that causes HF. Methods: The GSE5406 database was used for screening the differentially expressed genes (DEGs). Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Protein-Protein Interaction (PPI) network were applied to analyze DEGs. Besides, cell counting Kit-8 (CCK-8) was conducted to observe the knockdown effect of hub genes on cell proliferation. Results: Finally, 377 upregulated and 461 downregulated DEGs came out, enriched in the extracellular matrix organization and gap junction. According to GSEA results, Hoft cd4 positive alpha beta memory t cell bcg vaccine age 18-45 yo id 7 dy top 100 deg ex vivo up, Sobolev t cell pandemrix age 18-64 yo 7 dy dn, and so on were significantly related to gene set GSE5406. 7 hub genes, such as COL1A1, UBB, COL3A1, HSP90AA1, MYC, STAT3 and MAPK1, were selected from PPI networks. CCK-8 indicated silencing of STAT3 promoted the proliferation of H9C2 cells and silencing of UBB inhibited the proliferation of H9C2 cells. Conclusion: Our analysis reveals that COL1A1, UBB, COL3A1, HSP90AA1, MYC, STAT3, and MAPK1 might promote the progression of HF and become the biomarkers for diagnosis and treatment of HF.

The dynamic N(1)-methyladenosine methylome in eukaryotic messenger RNA.

Gene expression can be regulated post-transcriptionally through dynamic and reversible RNA modifications. A recent noteworthy example is N(6)-methyladenosine (m(6)A), which affects messenger RNA (mRNA) localization, stability, translation and splicing. Here we report on a new mRNA modification, N(1)-methyladenosine (m(1)A), that occurs on thousands of different gene transcripts in eukaryotic cells, from yeast to mammals, at an estimated average transcript stoichiometry of 20% in humans. Employing newly developed sequencing approaches, we show that m(1)A is enriched around the start codon upstream of the first splice site: it preferentially decorates more structured regions around canonical and alternative translation initiation sites, is dynamic in response to physiological conditions, and correlates positively with protein production. These unique features are highly conserved in mouse and human cells, strongly indicating a functional role for m(1)A in promoting translation of methylated mRNA.

Influence of Gut Microbiota and Trimethylamine N-Oxide in Patients with Coronary Heart Disease.

In the current study, the gut microbiota of patients with and without coronary heart disease was compared and the relationship between gut microbiota distribution, intending to reveal the role of gut microbiota in the coronary atherosclerosis process, was investigated.This study included 50 patients diagnosed with coronary heart disease (CHD) who received conventional coronary angiography or computed tomography angiography and 50 patients with CHD at Changshu No. 2 People's Hospital, Suzhou, China, from May 2020 to January 2021. Trimethylamine N-oxide (TMAO) level was tested and feces were collected, the DNA of the gut microbiota was extracted, and the distribution by 16SrRNA gene sequencing was obtained from the two groups of patients.Plasma TMAO concentrations were significantly higher in patients with CHD (P < 0.001). In the CHD group, 22 patients with multivessel disease had a higher level of TMAO compared with the 28 patients who had the single-vessel disease (P < 0.001). No difference in the gut microbiota diversity was noted between the two groups (P < 0.001). Patients with CHD had a significantly lower proportion of Bacteroidetes phyla and more proportion of Epsilonbacteraeota phyla. At the genus level, patients with CHD had an increased abundance of Enterococcus, whereas healthy controls had significantly higher levels of Streptococcus. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States 2 analysis found that, in the KEGG ORTHOLOGY, the level of choline trimethylamine-lyase gene expression correlated with TMAO production was higher in the fecal microbiome of the CHD group (P < 0.05).Gut microbiota and its product were expected to become a diagnostic marker and a new target for preventing CHD.

N(6)-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions.

RNA-binding proteins control many aspects of cellular biology through binding single-stranded RNA binding motifs (RBMs). However, RBMs can be buried within their local RNA structures, thus inhibiting RNA-protein interactions. N(6)-methyladenosine (m(6)A), the most abundant and dynamic internal modification in eukaryotic messenger RNA, can be selectively recognized by the YTHDF2 protein to affect the stability of cytoplasmic mRNAs, but how m(6)A achieves its wide-ranging physiological role needs further exploration. Here we show in human cells that m(6)A controls the RNA-structure-dependent accessibility of RBMs to affect RNA-protein interactions for biological regulation; we term this mechanism 'the m(6)A-switch'. We found that m(6)A alters the local structure in mRNA and long non-coding RNA (lncRNA) to facilitate binding of heterogeneous nuclear ribonucleoprotein C (HNRNPC), an abundant nuclear RNA-binding protein responsible for pre-mRNA processing. Combining photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) and anti-m(6)A immunoprecipitation (MeRIP) approaches enabled us to identify 39,060 m(6)A-switches among HNRNPC-binding sites; and global m(6)A reduction decreased HNRNPC binding at 2,798 high-confidence m(6)A-switches. We determined that these m(6)A-switch-regulated HNRNPC-binding activities affect the abundance as well as alternative splicing of target mRNAs, demonstrating the regulatory role of m(6)A-switches on gene expression and RNA maturation. Our results illustrate how RNA-binding proteins gain regulated access to their RBMs through m(6)A-dependent RNA structural remodelling, and provide a new direction for investigating RNA-modification-coded cellular biology.

ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility.

N(6)-methyladenosine (m(6)A) is the most prevalent internal modification of messenger RNA (mRNA) in higher eukaryotes. Here we report ALKBH5 as another mammalian demethylase that oxidatively reverses m(6)A in mRNA in vitro and in vivo. This demethylation activity of ALKBH5 significantly affects mRNA export and RNA metabolism as well as the assembly of mRNA processing factors in nuclear speckles. Alkbh5-deficient male mice have increased m(6)A in mRNA and are characterized by impaired fertility resulting from apoptosis that affects meiotic metaphase-stage spermatocytes. In accordance with this defect, we have identified in mouse testes 1,551 differentially expressed genes that cover broad functional categories and include spermatogenesis-related mRNAs involved in the p53 functional interaction network. The discovery of this RNA demethylase strongly suggests that the reversible m(6)A modification has fundamental and broad functions in mammalian cells.

Determination of tRNA aminoacylation levels by high-throughput sequencing.

Transfer RNA (tRNA) decodes mRNA codons when aminoacylated (charged) with an amino acid at its 3' end. Charged tRNAs turn over rapidly in cells, and variations in charged tRNA fractions are known to be a useful parameter in cellular responses to stress. tRNA charging fractions can be measured for individual tRNA species using acid denaturing gels, or comparatively at the genome level using microarrays. These hybridization-based approaches cannot be used for high resolution analysis of mammalian tRNAs due to their large sequence diversity. Here we develop a high-throughput sequencing method that enables accurate determination of charged tRNA fractions at single-base resolution (Charged DM-tRNA-seq). Our method takes advantage of the recently developed DM-tRNA-seq method, but includes additional chemical steps that specifically remove the 3'A residue in uncharged tRNA. Charging fraction is obtained by counting the fraction of A-ending reads versus A+C-ending reads for each tRNA species in the same sequencing reaction. In HEK293T cells, most cytosolic tRNAs are charged at >80% levels, whereas tRNASer and tRNAThr are charged at lower levels. These low charging levels were validated using acid denaturing gels. Our method should be widely applicable for investigations of tRNA charging as a parameter in biological regulation.

Efficient and quantitative high-throughput tRNA sequencing.

Despite its biological importance, tRNA has not been adequately sequenced by standard methods because of its abundant post-transcriptional modifications and stable structure, which interfere with cDNA synthesis. We achieved efficient and quantitative tRNA sequencing in HEK293T cells by using engineered demethylases to remove base methylations and a highly processive thermostable group II intron reverse transcriptase to overcome these obstacles. Our method, DM-tRNA-seq, should be applicable to investigations of tRNA in all organisms.

tRNA base methylation identification and quantification via high-throughput sequencing.

Eukaryotic transfer RNAs contain on average 14 modifications. Investigations of their biological functions require the determination of the modification sites and the dynamic variations of the modification fraction. Base methylation represents a major class of tRNA modification. Although many approaches have been used to identify tRNA base methylations, including sequencing, they are generally qualitative and do not report the information on the modification fraction. Dynamic mRNA modifications have been shown to play important biological roles; yet, the extent of tRNA modification fractions has not been reported systemically. Here we take advantage of a recently developed high-throughput sequencing method (DM-tRNA-seq) to identify and quantify tRNA base methylations located at the Watson-Crick face in HEK293T cells at single base resolution. We apply information derived from both base mutations and positional stops from sequencing using a combination of demethylase treatment and cDNA synthesis by a thermophilic reverse transcriptase to compile a quantitative "Modification Index" (MI) for six base methylations in human tRNA and rRNA. MI combines the metrics for mutational and stop components from alignment of sequencing data without demethylase treatment, and the modifications are validated in the sequencing data upon demethylase treatment. We identify many new methylation sites in both human nuclear and mitochondrial-encoded tRNAs not present in the RNA modification databases. The potentially quantitative nature of the MI values obtained from sequencing is validated by primer extension of several tRNAs. Our approach should be widely applicable to identify tRNA methylation sites, analyze comparative fractional modifications, and evaluate the modification dynamics between different samples.

Liver kinase B1 suppresses growth of lung cancer cells through sonic hedgehog signaling pathway.

Lung cancer is one of life-threatening cancers in the worldwide. Liver kinase B1 (LKB1) has been reported to be closely related to cancers; however, the underlying mechanism of LKB1 in lung cancer remains unclear. In our study, a LKB1 specific shRNA was employed to down-regulate LKB1 levels and a LKB1 over-expression plasmid was constructed to up-regulate LKB1 levels. Thereafter, growth of lung cancer cells was assessed by MTT assay and flow cytometry. Effects of LKB1 on the activation of sonic hedgehog (Shh) signaling pathway were detected by Western blot. Effects of LKB1 on lung cancer growth and Shh signaling pathway activation were also assessed in vivo. Our results showed that LKB1 inhibited proliferation of lung cancer cells and induced their apoptosis. Moreover, LKB1 inhibited Shh signaling pathway activation. Our in vivo study also showed that LKB1 inhibited lung cancer growth in vivo and modulated Shh signaling pathway. Treatment with cyclopamine, a Shh signaling pathway inhibitor, reversed the effects of LKB1 silencing and enhanced the effects of LKB1 over-expression. Results of our study demonstrate that LKB1 inhibits lung cancer growth in vitro and in vivo through Shh signaling pathway.

Dynamics of spontaneous flipping of a mismatched base in DNA duplex.

DNA base flipping is a fundamental theme in DNA biophysics. The dynamics for a B-DNA base to spontaneously flip out of the double helix has significant implications in various DNA-protein interactions but are still poorly understood. The spontaneous base-flipping rate obtained previously via the imino proton exchange assay is most likely the rate of base wobbling instead of flipping. Using the diffusion-decelerated fluorescence correlation spectroscopy together with molecular dynamics simulations, we show that a base of a single mismatched base pair (T-G, T-T, or T-C) in a double-stranded DNA can spontaneously flip out of the DNA duplex. The extrahelical lifetimes are on the order of 10 ms, whereas the intrahelical lifetimes range from 0.3 to 20 s depending on the stability of the base pairs. These findings provide detailed understanding on the dynamics of DNA base flipping and lay down foundation to fully understand how exactly the repair proteins search and locate the target mismatched base among a vast excess of matched DNA bases.

Selective Enzymatic Demethylation of N2 ,N2 -Dimethylguanosine in RNA and Its Application in High-Throughput tRNA Sequencing.

The abundant Watson-Crick face methylations in biological RNAs such as N1 -methyladenosine (m1 A), N1 -methylguanosine (m1 G), N3 -methylcytosine (m3 C), and N2 ,N2 -dimethylguanosine (m22 G) cause significant obstacles for high-throughput RNA sequencing by impairing cDNA synthesis. One strategy to overcome this obstacle is to remove the methyl group on these modified bases prior to cDNA synthesis using enzymes. The wild-type E. coli AlkB and its D135S mutant can remove most of m1 A, m1 G, m3 C modifications in transfer RNA (tRNA), but they work poorly on m22 G. Here we report the design and evaluation of a series of AlkB mutants against m22 G-containing model RNA substrates that we synthesize using an improved synthetic method. We show that the AlkB D135S/L118V mutant efficiently and selectively converts m22 G modification to N2 -methylguanosine (m2 G). We also show that this new enzyme improves the efficiency of tRNA sequencing.

Iron-catalysed oxidation intermediates captured in a DNA repair dioxygenase.

Mononuclear iron-containing oxygenases conduct a diverse variety of oxidation functions in biology, including the oxidative demethylation of methylated nucleic acids and histones. Escherichia coli AlkB is the first such enzyme that was discovered to repair methylated nucleic acids, which are otherwise cytotoxic and/or mutagenic. AlkB human homologues are known to play pivotal roles in various processes. Here we present structural characterization of oxidation intermediates for these demethylases. Using a chemical cross-linking strategy, complexes of AlkB-double stranded DNA (dsDNA) containing 1,N(6)-etheno adenine (εA), N(3)-methyl thymine (3-meT) and N(3)-methyl cytosine (3-meC) are stabilized and crystallized, respectively. Exposing these crystals, grown under anaerobic conditions containing iron(II) and α-ketoglutarate (αKG), to dioxygen initiates oxidation in crystallo. Glycol (from εA) and hemiaminal (from 3-meT) intermediates are captured; a zwitterionic intermediate (from 3-meC) is also proposed, based on crystallographic observations and computational analysis. The observation of these unprecedented intermediates provides direct support for the oxidative demethylation mechanism for these demethylases. This study also depicts a general mechanistic view of how a methyl group is oxidatively removed from different biological substrates.

Probing N6-methyladenosine RNA modification status at single nucleotide resolution in mRNA and long noncoding RNA.

N(6)-methyladenosine (m(6)A) is the most abundant modification in mammalian mRNA and long noncoding RNA (lncRNA). Recent discoveries of two m(6)A demethylases and cell-type and cell-state-dependent m(6)A patterns indicate that m(6)A modifications are highly dynamic and likely play important biological roles for RNA akin to DNA methylation or histone modification. Proposed functions for m(6)A modification include mRNA splicing, export, stability, and immune tolerance; but m(6)A studies have been hindered by the lack of methods for its identification at single nucleotide resolution. Here, we develop a method that accurately determines m(6)A status at any site in mRNA/lncRNA, termed site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and thin-layer chromatography (SCARLET). The method determines the precise location of the m(6)A residue and its modification fraction, which are crucial parameters in probing the cellular dynamics of m(6)A modification. We applied the method to determine the m(6)A status at several sites in two human lncRNAs and three human mRNAs and found that m(6)A fraction varies between 6% and 80% among these sites. We also found that many m(6)A candidate sites in these RNAs are however not modified. The precise determination of m(6)A status in a long noncoding RNA also enables the identification of an m(6)A-containing RNA structural motif.

N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO.

We report here that fat mass and obesity-associated protein (FTO) has efficient oxidative demethylation activity targeting the abundant N6-methyladenosine (m(6)A) residues in RNA in vitro. FTO knockdown with siRNA led to increased amounts of m(6)A in mRNA, whereas overexpression of FTO resulted in decreased amounts of m(6)A in human cells. We further show the partial colocalization of FTO with nuclear speckles, which supports the notion that m(6)A in nuclear RNA is a major physiological substrate of FTO.

Sprouts of RNA epigenetics: the discovery of mammalian RNA demethylases.

More than 100 structurally distinct RNA modifications have been identified in all kingdoms of life. These post-transcriptional modifications are widely present in various RNAs, including ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA), long non-coding RNA (lncRNA), etc. We have shown that the methylation of N(6)-methyladenine (m(6)A) can be reversed through the discovery of the first RNA demethylase, the human fat mass and obesity-associated protein, FTO, in 2011. (Most recently, we have identified a new mammalian RNA demethylase, ALKBH5, which is also able to remove the methyl group of m(6)A from RNA both in vitro and in vivo (Fig. 1A). The ALKBH5 protein colocalizes with nuclear speckles where pre-mRNA processing occurs. This protein is actively involved in mRNA export regulation, in which its demethylation activity seems to play an important role, as well as in RNA synthesis. A knockout of the Alkbh5 gene in mice resulted in impaired male fertility due to compromised spermatogenesis. Importantly, increased m(6)A levels were observed in mRNA isolated from the Alkbh5-knockout mouse organs compared to those from wild-type littermates. RNA-Seq results indicate aberrant gene expression in spermatogenic cells of the seminoferous tubulus of testes from Alkbh5-deficient mice, thereby showing that the loss of the m(6)A demethylase influences gene expression, which, in turn, leads to defects in spermatogenesis and increased apoptosis of meiotic cells. Thus, the discovery of FTO and this new RNA demethylase strongly suggests that the methylation of RNA, like DNA and histone modifications, is dynamically regulated and likely to play broad roles in mammalian cells.

Structure determination of DNA methylation lesions N1-meA and N3-meC in duplex DNA using a cross-linked protein-DNA system.

N(1)-meA and N(3)-meC are cytotoxic DNA base methylation lesions that can accumulate in the genomes of various organisms in the presence of S(N)2 type methylating agents. We report here the structural characterization of these base lesions in duplex DNA using a cross-linked protein-DNA crystallization system. The crystal structure of N(1)-meA:T pair shows an unambiguous Hoogsteen base pair with a syn conformation adopted by N(1)-meA, which exhibits significant changes in the opening, roll and twist angles as compared to the normal A:T base pair. Unlike N(1)-meA, N(3)-meC does not establish any interaction with the opposite G, but remains partially intrahelical. Also, structurally characterized is the N(6)-meA base modification that forms a normal base pair with the opposite T in duplex DNA. Structural characterization of these base methylation modifications provides molecular level information on how they affect the overall structure of duplex DNA. In addition, the base pairs containing N(1)-meA or N(3)-meC do not share any specific characteristic properties except that both lesions create thermodynamically unstable regions in a duplex DNA, a property that may be explored by the repair proteins to locate these lesions.

Kinetic gating mechanism of DNA damage recognition by Rad4/XPC.

The xeroderma pigmentosum C (XPC) complex initiates nucleotide excision repair by recognizing DNA lesions before recruiting downstream factors. How XPC detects structurally diverse lesions embedded within normal DNA is unknown. Here we present a crystal structure that captures the yeast XPC orthologue (Rad4) on a single register of undamaged DNA. The structure shows that a disulphide-tethered Rad4 flips out normal nucleotides and adopts a conformation similar to that seen with damaged DNA. Contrary to many DNA repair enzymes that can directly reject non-target sites as structural misfits, our results suggest that Rad4/XPC uses a kinetic gating mechanism whereby lesion selectivity arises from the kinetic competition between DNA opening and the residence time of Rad4/XPC per site. This mechanism is further supported by measurements of Rad4-induced lesion-opening times using temperature-jump perturbation spectroscopy. Kinetic gating may be a general mechanism used by site-specific DNA-binding proteins to minimize time-consuming interrogations of non-target sites.