A dynamic reversible RNA N6-methyladenosine modification: current status and perspectives

N⁶-methyladenosine (m⁶A), as the most abundant RNA epigenetic modifications, has been shown to play critical roles in various biological functions. Research about enzymes that can catalyze and remove m⁶A have revealed its comprehensive roles in messenger RNA (mRNA) metabolism and other physiological processes. The “readers” including YTH domain-containing proteins, hnRNPC, hnRNPG, hnRNPA2B1, IGF2BP1, IGF2BP2, and IGF2BP3, which can affect the fates of mRNA in an m⁶A-dependent manner. In this review, we focus on recent advances in the research of the m⁶A modifications, especially about the latest functions of its writers, erasers, readers in RNA metabolism, cancer, and lipid metabolism. In the end, we provide insights into the underlying molecular mechanisms of m⁶A modifications.     

Modifications in small interfering RNA that separate immunostimulation from RNA interference

Synthetic small interfering RNA (siRNA) can suppress the expression of endogenous mRNA through RNA interference. It has been reported that siRNA can induce type I IFN production from plasmacytoid dendritic cells, leading to off-target effects. To separate immunostimulation from the desired gene-specific inhibitory activity, we designed RNA strands with chemical modifications at strategic positions of the ribose or nucleobase residues. Substitution of uridine residues by 2'-deoxyuridine or thymidine residues was found to decrease type I IFN production upon in vitro stimulation of human PBMC. Thymidine residues in both strands of a siRNA duplex further decreased immunostimulation. Fortunately, the thymidine residues did not affect gene-silencing activity. In contrast, 2'-O-methyl groups at adenosine and uridine residues reduced both IFN-alpha secretion and gene-silencing activity. Oligoribonucleotides with 2'-O-methyladenosine residues actively inhibited IFN-alpha secretion induced by other immunostimulatory RNAs, an effect not observed for strands with 2'-deoxynucleosides. Furthermore, neither 5-methylcytidine nor 7-deazaguanosine residues in the stimulatory strands affected IFN-alpha secretion, suggesting that recognition does not involve sites in the major groove of duplex regions. The activity data, together with structure prediction and exploratory UV-melting analyses, suggest that immunostimulatory sequences adopt folded structures. The results show that immunostimulation can be suppressed by suitable chemical modifications without losing siRNA potency by introducing seemingly minor structural changes.     

Specific and non-specific mammalian RNA terminal uridylyl transferases

Uridylylation of various types of RNA molecules is a wide-spread phenomenon in molecular biology and is catalyzed by enzymes mediating the transfer of UMP residues to the 3'-ends of preexisting RNA. In most cases, however, the biological significance of these modifications remains elusive. As an exception, the RNA terminal uridylyl transferases (TUTases) of the mRNA editing complex within mitochondria of Trypanosomatidae have been characterized in great detail. Current knowledge on those editing enzymes has been summarized recently by R. Aphasizhev [Cell. Mol. Life Sci. 62 (2005) 2194-203] and, therefore, will not be included here. Rather, this review will focus on cellular non-editing TUTases, characterized by distinct modes of catalytic activity and substrate specificity. Putative biological functions of this rapidly growing number of RNA modifying enzymes are discussed.     

Analysis of RNA binding by the dengue virus NS5 RNA capping enzyme

Flaviviruses are small, capped positive sense RNA viruses that replicate in the cytoplasm of infected cells. Dengue virus and other related flaviviruses have evolved RNA capping enzymes to form the viral RNA cap structure that protects the viral genome and directs efficient viral polyprotein translation. The N-terminal domain of NS5 possesses the methyltransferase and guanylyltransferase activities necessary for forming mature RNA cap structures. The mechanism for flavivirus guanylyltransferase activity is currently unknown, and how the capping enzyme binds its diphosphorylated RNA substrate is important for deciphering how the flavivirus guanylyltransferase functions. In this report we examine how flavivirus NS5 N-terminal capping enzymes bind to the 5' end of the viral RNA using a fluorescence polarization-based RNA binding assay. We observed that the K(D) for RNA binding is approximately 200 nM Dengue, Yellow Fever, and West Nile virus capping enzymes. Removal of one or both of the 5' phosphates reduces binding affinity, indicating that the terminal phosphates contribute significantly to binding. RNA binding affinity is negatively affected by the presence of GTP or ATP and positively affected by S-adensyl methoninine (SAM). Structural superpositioning of the dengue virus capping enzyme with the Vaccinia virus VP39 protein bound to RNA suggests how the flavivirus capping enzyme may bind RNA, and mutagenesis analysis of residues in the putative RNA binding site demonstrate that several basic residues are critical for RNA binding. Several mutants show differential binding to 5' di-, mono-, and un-phosphorylated RNAs. The mode of RNA binding appears similar to that found with other methyltransferase enzymes, and a discussion of diphosphorylated RNA binding is presented.     

Biosynthesis of transfer RNA: isolation and characterization of precursors to transfer RNA in the posterior silkgland of Bombyx mori.

Distinct low molecular weight RNA species that have properties expected for the precursor to tRNA have been isolated from the posterior silkglands of the silkworm Bombyx mori. These RNAs migrate between 4 S and 5 S markers on acrylamide gels and are labeled preferentially in vivo in relation to tRNA. The precursor RNAs can be converted specifically into molecules indistinguishable in size from tRNA upon incubation with “cleavage” enzymes isolated from the silkgland ribosomes. Two of the three low molecular weight RNAs contain the modified residues, pseudouridine, dihydrouridine and ribothymidine, and are methylated in vivo, suggesting that these base modifications occur while the tRNA is still in its precursor stage.     

Synthesis of pyrophosphates for in vitro selection of catalytic RNA molecules

Reported here are synthetic routes to pyrophosphates linking riboflavin with various nucleosides. The focus is on a flavin-uracil dinucleotide having a biotin tag on the uracil, a molecule that has potential value in the selection of RNA enzymes that catalyze the template-directed polymerization of RNA in the 3'-to-5' direction, which is the direction opposite that catalyzed by standard protein polymerases. Two detailed procedures are presented to prepare this new compound, as well as one procedure to prepare the new flavin-2,6-diaminopurine dinucleotide.     

Escherichia coli 5S RNA A and B conformers. Characterisation by enzymatic and chemical methods

The structures of the two stable conformers of Escherichia coli 5 S RNA, the and B form, were compared. Information about the structures were obtained using the methods of limited enzymatic hydrolysis and chemical modification of accessible nucleotides. Base-specific modifications were performed for adenosines and cytidines using diethylpyrocarbonate and dimethylsulfate in combination with a strand-scission reaction at the modified site. Base-specific (RNase T1) as well as conformation-specific (nuclease S1, cobra venom nuclease) enzymes were employed for the limited enzymatic hydrolysis. Clear differences in the accessibility of the two 5 S RNA conformers to the enzymes and the chemical reagents were established and the regions with altered reactivities were localized in the 5 S RNA structure. The results are consistent with the disruption of the secondary structural interactions in helix II and partly in helices III and IV during the transition from the A to the B form. (The numbering of the helices is according to the generally accepted Fox and Woese model.) In addition some regions presumably involved in the tertiary structure are distorted. There is evidence, however, for the new formation of structural regions between two distant sites in the 5 S RNA B form. The results enable us to refine the existing 5 S RNA A-form model and provide insight into the structural dynamics that lead to the formation of the 5 S RNA B form.     

Focus on function: single molecule RNA enzymology

The ability of RNA to catalyze chemical reactions was first demonstrated 25 years ago with the discovery that group I introns and RNase P function as RNA enzymes (ribozymes). Several additional ribozymes were subsequently identified, most notably the ribosome, followed by intense mechanistic studies. More recently, the introduction of single molecule tools has dissected the kinetic steps of several ribozymes in unprecedented detail and has revealed surprising heterogeneity not evident from ensemble approaches. Still, many fundamental questions of how RNA enzymes work at the molecular level remain unanswered. This review surveys the current status of our understanding of RNA catalysis at the single molecule level and discusses the existing challenges and opportunities in developing suitable assays.     

NSUN6 is a human RNA methyltransferase that catalyzes formation of m5C72 in specific tRNAs

Many cellular RNAs require modification of specific residues for their biogenesis, structure, and function. 5-methylcytosine (m⁵C) is a common chemical modification in DNA and RNA but in contrast to the DNA modifying enzymes, only little is known about the methyltransferases that establish m⁵C modifications in RNA. The putative RNA methyltransferase NSUN6 belongs to the family of Nol1/Nop2/SUN domain (NSUN) proteins, but so far its cellular function has remained unknown. To reveal the target spectrum of human NSUN6, we applied UV crosslinking and analysis of cDNA (CRAC) as well as chemical crosslinking with 5-azacytidine. We found that human NSUN6 is associated with tRNAs and acts as a tRNA methyltransferase. Furthermore, we uncovered tRNA^Cys and tRNA^Thr as RNA substrates of NSUN6 and identified the cytosine C72 at the 3′ end of the tRNA acceptor stem as the target nucleoside. Interestingly, target recognition in vitro depends on the presence of the 3′-CCA tail. Together with the finding that NSUN6 localizes to the cytoplasm and largely colocalizes with marker proteins for the Golgi apparatus and pericentriolar matrix, our data suggest that NSUN6 modifies tRNAs in a late step in their biogenesis.     

The C-terminal carboxy group of T7 RNA polymerase ensures efficient magnesium ion-dependent catalysis.

DNA and RNA polymerases use divalent metal ions for catalysis. Crystal structures of several polymerases reveal that two acidic residues are involved in coordinating two metal ions at the catalytic centre. Bacteriophage RNA polymerases contain a highly conserved C-terminus with the carboxylate positioned near the active site. We examined whether theC-terminal carboxy group of T7 RNA polymerase is important for magnesium ion-dependent catalysis. Introduction of a methyl ester or decarboxylation of the C-terminal carboxy group was achieved with an intein-based protein expression system and an elongation rate assay was developed to test the effects of the modifications. The results show that enzymes with a modified C-terminal carboxy group exhibit a magnesium ion-dependent decrease in catalytic activity.     

RNA 3'-phosphate cyclase (RtcA) catalyzes ligase-like adenylylation of DNA and RNA 5'-monophosphate ends

RNA 3'-phosphate cyclase (Rtc) enzymes are a widely distributed family that catalyze the synthesis of RNA 2',3'-cyclic phosphate ends via an ATP-dependent pathway comprising three nucleotidyl transfer steps: reaction of Rtc with ATP to form a covalent Rtc-(histidinyl-N)-AMP intermediate and release PP(i); transfer of AMP from Rtc to an RNA 3'-phosphate to form an RNA(3')pp(5')A intermediate; and attack by the terminal nucleoside O2' on the 3'-phosphate to form an RNA 2',3'-cyclic phosphate product and release AMP. The chemical transformations of the cyclase pathway resemble those of RNA and DNA ligases, with the key distinction being that ligases covalently adenylylate 5'-phosphate ends en route to phosphodiester synthesis. Here we show that the catalytic repertoire of RNA cyclase overlaps that of ligases. We report that Escherichia coli RtcA catalyzes adenylylation of 5'-phosphate ends of DNA or RNA strands to form AppDNA and AppRNA products. The polynucleotide 5' modification reaction requires the His(309) nucleophile, signifying that it proceeds through a covalent RtcA-AMP intermediate. We established this point directly by demonstrating transfer of [(32)P]AMP from RtcA to a pDNA strand. RtcA readily adenylylated the 5'-phosphate at a 5'-PO(4)/3'-OH nick in duplex DNA but was unable to covert the nicked DNA-adenylate to a sealed phosphodiester. Our findings raise the prospect that cyclization of RNA 3'-ends might not be the only biochemical pathway in which Rtc enzymes participate; we discuss scenarios in which the 5'-adenylyltransferase of RtcA might play a role.     

Mechanism of U-Insertion RNA Editing in Trypanosome Mitochondria: Characterization of RET2 Functional Domains by Mutational Analysis

3′-Terminal uridylyl transferases (TUTases) selectively bind uridine 5′-triphosphate (UTP) and catalyze the addition of uridine 5′-monophosphate to the 3′-hydroxyl of RNA substrates in a template-independent manner. RNA editing TUTase 1 and RNA editing TUTase 2 (RET2) play central roles in uridine insertion/deletion RNA editing, which is an essential part of mitochondrial RNA processing in trypanosomes. Although the conserved N-terminal (catalytic) domain and C-terminal (nucleotide base recognition) domain are readily distinguished in all known TUTases, nucleotide specificity, RNA substrate preference, processivity, quaternary structures, and auxiliary domains vary significantly among enzymes of divergent biological functions. RET2 acts as a subunit of the RNA editing core complex to carry out guide-RNA-dependent U-insertion into mitochondrial mRNA. By correlating mutational effects on RET2 activity as recombinant protein and as RNA editing core complex subunit with RNAi-based knock-in phenotypes, we have assessed the UTP and RNA binding sites in RET2. Here we demonstrate functional conservation of key UTP-binding and metal-ion-coordinating residues and identify amino acids involved in RNA substrate recognition. Invariant arginine residues 144 and 435 positioned in the vicinity of the UTP binding site are critical for RET2 activity on single-stranded and double-stranded RNAs, as well as function in vivo. Recognition of a double-stranded RNA, which resembles a guide RNA/mRNA duplex, is further facilitated by multipoint contacts across the RET2-specific middle domain.     

RNA甲基化修饰调控和规律

表观遗传学修饰包括DNA、RNA和蛋白质的化学修饰,基于非序列改变所致基因表达和功能水平变化。近年来,在DNA和蛋白质修饰基础上,可逆RNA甲基化修饰研究引领了第3次表观遗传学修饰研究的浪潮。RNA存在100余种化学修饰,甲基化是最主要的修饰形式。鉴定RNA甲基化修饰酶及研发其转录组水平高通量检测技术,是揭示RNA化学修饰调控基因表达和功能规律的基础。本文主要总结了近年来本课题组与合作团队及国内外同行在RNA甲基化表观转录组学研究中取得的主要前沿进展,包括发现了RNA去甲基酶、甲基转移酶和结合蛋白,揭示RNA甲基化修饰调控RNA加工代谢,及其调控正常生理和异常病理等重要生命进程。这些系列研究成果证明RNA甲基化修饰类似于DNA甲基化,具有可逆性,拓展了RNA甲基化表观转录组学研究新领域,完善了中心法则表观遗传学规律。     

Novel role for RNase PH in the degradation of structured RNA

Escherichia coli contains multiple 3' to 5' RNases, of which two, RNase PH and polynucleotide phosphorylase (PNPase), use inorganic phosphate as a nucleophile to catalyze RNA cleavage. It is known that an absence of these two enzymes causes growth defects, but the basis for these defects has remained undefined. To further an understanding of the function of these enzymes, the degradation pattern of different cellular RNAs was analyzed. It was observed that an absence of both enzymes results in the appearance of novel mRNA degradation fragments. Such fragments were also observed in strains containing mutations in RNase R and PNPase, enzymes whose collective absence is known to cause an accumulation of structured RNA fragments. Additional experiments indicated that the growth defects of strains containing RNase R and PNPase mutations were exacerbated upon RNase PH removal. Taken together, these observations suggested that RNase PH could play a role in structured RNA degradation. Biochemical experiments with RNase PH demonstrated that this enzyme digests through RNA duplexes of moderate stability. In addition, mapping and sequence analysis of an mRNA degradation fragment that accumulates in the absence of the phosphorolytic enzymes revealed the presence of an extended stem-loop motif at the 3' end. Overall, these results indicate that RNase PH plays a novel role in the degradation of structured RNAs and provides a potential explanation for the growth defects caused by an absence of the phosphorolytic RNases.     

The polynucleotide ligase and RNA capping enzyme superfamily of covalent nucleotidyltransferases

ATP- and NAD(+)-dependent DNA ligases, ATP-dependent RNA ligases and GTP-dependent mRNA capping enzymes comprise a superfamily of proteins that catalyze nucleotidyl transfer to polynucleotide 5' ends via covalent enzyme-(lysyl-N)-NMP intermediates. The superfamily is defined by five peptide motifs that line the nucleotide-binding pocket and contribute amino acid sidechains essential for catalysis. Early crystal structures revealed a shared core tertiary structure for DNA ligases and capping enzymes, which are composed minimally of a nucleotidyltransferase domain fused to a distal OB-fold domain. Recent structures of viral and bacterial DNA ligases, and a fungal mRNA capping enzyme underscore how the substrate-binding and chemical steps of the ligation and capping pathways are coordinated with large rearrangements of the component protein domains and with remodeling of the atomic contacts between the enzyme and the nucleotide at the active site. The first crystal structure of an RNA ligase suggests that contemporary DNA ligases, RNA ligases and RNA capping enzymes evolved by fusion of ancillary effector domains to an ancestral catalytic module involved in RNA repair.