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

N⁶-methyladenosine (m⁶A) is the most abundant modification in mammalian mRNA and long noncoding RNA (lncRNA). Recent discoveries of two m⁶A demethylases and cell-type and cell-state-dependent m⁶A patterns indicate that m⁶A modifications are highly dynamic and likely play important biological roles for RNA akin to DNA methylation or histone modification. Proposed functions for m⁶A modification include mRNA splicing, export, stability, and immune tolerance; but m⁶A studies have been hindered by the lack of methods for its identification at single nucleotide resolution. Here, we develop a method that accurately determines m⁶A 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 m⁶A residue and its modification fraction, which are crucial parameters in probing the cellular dynamics of m⁶A modification. We applied the method to determine the m⁶A status at several sites in two human lncRNAs and three human mRNAs and found that m⁶A fraction varies between 6% and 80% among these sites. We also found that many m⁶A candidate sites in these RNAs are however not modified. The precise determination of m⁶A status in a long noncoding RNA also enables the identification of an m⁶A-containing RNA structural motif.     

Crystal structure of the RNA demethylase ALKBH5 from zebrafish

ALKBH5, a member of AlkB family proteins, has been reported as a mammalian N⁶-methyladenosine (m⁶A) RNA demethylase. Here we report the crystal structure of zebrafish ALKBH5 (fALKBH5) with the resolution of 1.65 Å. Structural superimposition shows that fALKBH5 is comprised of a conserved jelly-roll motif. However, it possesses a loop that interferes potential binding of a duplex nucleic acid substrate, suggesting an important role in substrate selection. In addition, several active site residues are different between the two known m⁶A RNA demethylases, ALKBH5 and FTO, which may result in their slightly different pathways of m⁶A demethylation.     

O6-Methylguanosine leads to position-dependent effects on ribosome speed and fidelity

Nucleic acids are under constant assault from endogenous and environmental agents that alter their physical and chemical properties. O6-methylation of guanosine (m⁶G) is particularly notable for its high mutagenicity, pairing with T, during DNA replication. Yet, while m⁶G accumulates in both DNA and RNA, little is known about its effects on RNA. Here, we investigate the effects of m⁶G on the decoding process, using a reconstituted bacterial translation system. m⁶G at the first and third position of the codon decreases the accuracy of tRNA selection. The ribosome readily incorporates near-cognate aminoacyl-tRNAs (aa-tRNAs) by forming m⁶G-uridine codon–anticodon pairs. Surprisingly, the introduction of m⁶G to the second position of the codon does not promote miscoding, but instead slows the observed rates of peptide-bond formation by >1000-fold for cognate aa-tRNAs without altering the rates for near-cognate aa-tRNAs. These in vitro observations were recapitulated in eukaryotic extracts and HEK293 cells. Interestingly, the analogous modification N6-methyladenosine (m⁶A) at the second position has only a minimal effect on tRNA selection, suggesting that the effects on tRNA selection seen with m⁶G are due to altered geometry of the base pair. Given that the m6G:U base pair is predicted to be nearly indistinguishable from a Watson-Crick base pair, our data suggest that the decoding center of the ribosome is extremely sensitive to changes at the second position. Our data, apart from highlighting the deleterious effects that these adducts pose to cellular fitness, shed new insight into decoding and the process by which the ribosome recognizes codon–anticodon pairs.     

The detection and functions of RNA modification m6A based on m6A writers and erasers

N⁶-methyladenosine (m⁶A) is the most frequent chemical modification in eukaryotic mRNA and is known to participate in a variety of physiological processes, including cancer progression and viral infection. The reversible and dynamic m⁶A modification is installed by m⁶A methyltransferase (writer) enzymes and erased by m⁶A demethylase (eraser) enzymes. m⁶A modification recognized by m⁶A binding proteins (readers) regulates RNA processing and metabolism, leading to downstream biological effects such as promotion of stability and translation or increased degradation. The m⁶A writers and erasers determine the abundance of m⁶A modifications and play decisive roles in its distribution and function. In this review, we focused on m⁶A writers and erasers and present an overview on their known functions and enzymatic molecular mechanisms, showing how they recognize substrates and install or remove m⁶A modifications. We also summarize the current applications of m⁶A writers and erasers for m⁶A detection and highlight the merits and drawbacks of these available methods. Lastly, we describe the biological functions of m⁶A in cancers and viral infection based on research of m⁶A writers and erasers and introduce new assays for m⁶A functionality via programmable m⁶A editing tools.     

YccW is the m5C methyltransferase specific for 23S rRNA nucleotide 1962

Methylation at the 5-position of cytosine [m⁵C (5-methylcytidine)] occurs at three RNA nucleotides in Escherichia coli. All these modifications are at highly conserved nucleotides in the rRNAs, and each is catalyzed by its own m⁵C methyltransferase enzyme. Two of the enzymes, RsmB and RsmF, are already known and methylate 16S rRNA at nucleotides C967 and C1407, respectively. Here, we report the identity of the third E. coli m⁵C methyltransferase. Analysis of rRNAs by matrix-assisted laser desorption/ionization mass spectrometry showed that inactivation of the yccW gene leads to loss of m⁵C methylation at nucleotide 1962 in E. coli 23S rRNA. This methylation is restored by complementing the knockout strain with a plasmid-encoded copy of the yccW gene. Purified recombinant YccW protein retains its specificity for C1962 in vitro and methylates naked 23S rRNA isolated from the yccW knockout strain. However, YccW does not methylate assembled 50S subunits, and this is somewhat surprising as the published crystal structures show nucleotide C1962 to be fully accessible at the subunit interface. YccW-directed methylation at nucleotide C1962 is conserved in bacteria, and loss of this methylation in E. coli marginally reduces its growth rate. YccW had previously eluded identification because it displays only limited sequence similarity to the m⁵C methyltransferases RsmB and RsmF and is in fact more similar to known m⁵U (5-methyluridine) RNA methyltransferases. In keeping with the previously proposed nomenclature system for bacterial rRNA methyltransferases, yccW is now designated as the rRNA large subunit methyltransferase gene rlmI.     

The METTL5-TRMT112 N6-methyladenosine methyltransferase complex regulates mRNA translation via 18S rRNA methylation

Ribosomal RNAs (rRNAs) have long been known to carry chemical modifications, including 2′O-methylation, pseudouridylation, N⁶-methyladenosine (m⁶A), and N^6,6-dimethyladenosine. While the functions of many of these modifications are unclear, some are highly conserved and occur in regions of the ribosome critical for mRNA decoding. Both 28S rRNA and 18S rRNA carry single m⁶A sites, and while the methyltransferase ZCCHC4 has been identified as the enzyme responsible for the 28S rRNA m⁶A modification, the methyltransferase responsible for the 18S rRNA m⁶A modification has remained unclear. Here, we show that the METTL5-TRMT112 methyltransferase complex installs the m⁶A modification at position 1832 of human 18S rRNA. Our work supports findings that TRMT112 is required for METTL5 stability and reveals that human METTL5 mutations associated with microcephaly and intellectual disability disrupt this interaction. We show that loss of METTL5 in human cancer cell lines and in mice regulates gene expression at the translational level; additionally, Mettl5 knockout mice display reduced body size and evidence of metabolic defects. While recent work has focused heavily on m⁶A modifications in mRNA and their roles in mRNA processing and translation, we demonstrate here that deorphanizing putative methyltransferase enzymes can reveal previously unappreciated regulatory roles for m⁶A in noncoding RNAs.     

Intrinsic resistance to aminoglycosides in Enterococcus faecium is conferred by the 16S rRNA m5C1404-specific methyltransferase EfmM

Aminoglycosides are ribosome-targeting antibiotics and a major drug group of choice in the treatment of serious enterococcal infections. Here we show that aminoglycoside resistance in Enterococcus faecium strain CIP 54-32 is conferred by the chromosomal gene efmM, encoding the E. faecium methyltransferase, as well as by the previously characterized aac(6′)-Ii that encodes a 6′-N-aminoglycoside acetyltransferase. Inactivation of efmM in E. faecium increases susceptibility to the aminoglycosides kanamycin and tobramycin, and, conversely, expression of a recombinant version of efmM in Escherichia coli confers resistance to these drugs. The EfmM protein shows significant sequence similarity to E. coli RsmF (previously called YebU), which is a 5-methylcytidine (m⁵C) methyltransferase modifying 16S rRNA nucleotide C1407. The target for EfmM is shown by mass spectrometry to be a neighboring 16S rRNA nucleotide at C1404. EfmM uses the methyl group donor S-adenosyl-L-methionine to catalyze formation of m⁵C1404 on the 30S ribosomal subunit, whereas naked 16S rRNA and the 70S ribosome are not substrates. Addition of the 5-methyl to C1404 sterically hinders aminoglycoside binding. Crystallographic structure determination of EfmM at 2.28 Å resolution reveals an N-terminal domain connected to a central methyltransferase domain that is linked by a flexible lysine-rich region to two C-terminal subdomains. Mutagenesis of the methyltransferase domain established that two cysteines at specific tertiary locations are required for catalysis. The tertiary structure of EfmM is highly similar to that of RsmF, consistent with m⁵C formation at adjacent sites on the 30S subunit, while distinctive structural features account for the enzymes'' respective specificities for nucleotides C1404 and C1407.     

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.     

Functional characterization of the Drosophila MRP (mitochondrial RNA processing) RNA gene

MRP RNA is a noncoding RNA component of RNase mitochondrial RNA processing (MRP), a multi-protein eukaryotic endoribonuclease reported to function in multiple cellular processes, including ribosomal RNA processing, mitochondrial DNA replication, and cell cycle regulation. A recent study predicted a potential Drosophila ortholog of MRP RNA (CR33682) by computer-based genome analysis. We have confirmed the expression of this gene and characterized the phenotype associated with this locus. Flies with mutations that specifically affect MRP RNA show defects in growth and development that begin in the early larval period and end in larval death during the second instar stage. We present several lines of evidence demonstrating a role for Drosophila MRP RNA in rRNA processing. The nuclear fraction of Drosophila MRP RNA localizes to the nucleolus. Further, a mutant strain shows defects in rRNA processing that include a defect in 5.8S rRNA processing, typical of MRP RNA mutants in other species, as well as defects in early stages of rRNA processing.     

The impact of the glial spatial buffering on the K+ Nernst potential

Astrocytes play a critical role in CNS metabolism, regulation of volume and ion homeostasis of the interstitial space. Of special relevance is their clearance of K⁺ that is released by active neurons into the extracellular space. Mathematical analysis of a modified Nernst equation for the electrochemical equilibrium of neuronal plasma membranes, suggests that K⁺ uptake by glial cells is not only relevant during neuronal activity but also has a non-neglectable impact on the basic electrical membrane properties, specifically the resting membrane potential, of neurons and might be clinically valuable as a factor in the genetics and epigenetics of the epilepsy and tuberous sclerosis complex.