Terminal Monophosphate Research Articles

Structure of the human DICER-pre-miRNA complex in a dicing state.

Dicer has a key role in small RNA biogenesis, processing double-stranded RNAs (dsRNAs)1,2. Human DICER (hDICER, also known as DICER1) is specialized for cleaving small hairpin structures such as precursor microRNAs (pre-miRNAs) and has limited activity towards long dsRNAs-unlike its homologues in lower eukaryotes and plants, which cleave long dsRNAs. Although the mechanism by which long dsRNAs are cleaved has been well documented, our understanding of pre-miRNA processing is incomplete because structures of hDICER in a catalytic state are lacking. Here we report the cryo-electron microscopy structure of hDICER bound to pre-miRNA in a dicing state and uncover the structural basis of pre-miRNA processing. hDICER undergoes large conformational changes to attain the active state. The helicase domain becomes flexible, which allows the binding of pre-miRNA to the catalytic valley. The double-stranded RNA-binding domain relocates and anchors pre-miRNA in a specific position through both sequence-independent and sequence-specific recognition of the newly identified 'GYM motif'3. The DICER-specific PAZ helix is also reoriented to accommodate the RNA. Furthermore, our structure identifies a configuration of the 5' end of pre-miRNA inserted into a basic pocket. In this pocket, a group of arginine residues recognize the 5' terminal base (disfavouring guanine) and terminal monophosphate; this explains the specificity of hDICER and how it determines the cleavage site. We identify cancer-associated mutations in the 5' pocket residues that impair miRNA biogenesis. Our study reveals how hDICER recognizes pre-miRNAs with stringent specificity and enables a mechanistic understanding of hDICER-related diseases.

Obstacles to Scanning by RNase E Govern Bacterial mRNA Lifetimes by Hindering Access to Distal Cleavage Sites

The diversity of mRNA lifetimes in bacterial cells is difficult to reconcile with the relaxed cleavage site specificity of RNase E, the endonuclease most important for governing mRNA degradation. This enzyme has generally been thought to locate cleavage sites by searching freely in three dimensions. However, our results now show that its access to such sites in 5'-monophosphorylated RNA is hindered by obstacles-such as bound proteins or ribosomes or coaxial small RNA (sRNA) base pairing-that disrupt the path from the 5' end to those sites and prolong mRNA lifetimes. These findings suggest that RNase E searches for cleavage sites by scanning linearly from the 5'-terminal monophosphate along single-stranded regions of RNA and that its progress is impeded by structural discontinuities encountered along the way. This discovery has major implications for gene regulation in bacteria and suggests a general mechanism by which other prokaryotic and eukaryotic regulatory proteins can be controlled.

Decreased Expression of Stable RNA Can Alleviate the Lethality Associated with RNase E Deficiency in Escherichia coli.

The endoribonuclease RNase E participates in mRNA degradation, rRNA processing, and tRNA maturation in Escherichia coli, but the precise reasons for its essentiality are unclear and much debated. The enzyme is most active on RNA substrates with a 5'-terminal monophosphate, which is sensed by a domain in the enzyme that includes residue R169; E. coli also possesses a 5'-pyrophosphohydrolase, RppH, that catalyzes conversion of 5'-terminal triphosphate to 5'-terminal monophosphate on RNAs. Although the C-terminal half (CTH), beyond residue approximately 500, of RNase E is dispensable for viability, deletion of the CTH is lethal when combined with an R169Q mutation or with deletion of rppH In this work, we show that both these lethalities can be rescued in derivatives in which four or five of the seven rrn operons in the genome have been deleted. We hypothesize that the reduced stable RNA levels under these conditions minimize the need of RNase E to process them, thereby allowing for its diversion for mRNA degradation. In support of this hypothesis, we have found that other conditions that are known to reduce stable RNA levels also suppress one or both lethalities: (i) alterations in relA and spoT, which are expected to lead to increased basal ppGpp levels; (ii) stringent rpoB mutations, which mimic high intracellular ppGpp levels; and (iii) overexpression of DksA. Lethality suppression by these perturbations was RNase R dependent. Our work therefore suggests that its actions on the various substrates (mRNA, rRNA, and tRNA) jointly contribute to the essentiality of RNase E in E. coliIMPORTANCE The endoribonuclease RNase E is essential for viability in many Gram-negative bacteria, including Escherichia coli Different explanations have been offered for its essentiality, including its roles in global mRNA degradation or in the processing of several tRNA and rRNA species. Our work suggests that, rather than its role in the processing of any one particular substrate, its distributed functions on all the different substrates (mRNA, rRNA, and tRNA) are responsible for the essentiality of RNase E in E. coli.

The multifunctional RNase XRN2

Different classes of RNA function in various cellular processes, and their biogenesis and turnover involve diverse RNases for processing and degradation. XRN2 is a 5'→3' exoribonuclease that is evolutionarily conserved in eukaryotes. It is predominantly localized in the nucleus and recognizes single-stranded RNA with a 5'-terminal monophosphate to degrade it processively to mononucleotides. In the present paper, we review functions of XRN2 and its cofactors in maturation, surveillance and activity control of several classes of RNA such as pre-mRNA (precursor mRNA), rRNA and snoRNA (small nucleolar RNA).

Bacteriophage T4 polynucleotide kinase triggers degradation of mRNAs

The bacteriophage T4-encoded RegB endoribonuclease is produced during the early stage of phage development and targets mostly (but not exclusively) the Shine-Dalgarno sequences of early genes. In this work, we show that the degradation of RegB-cleaved mRNAs depends on a functional T4 polynucleotide kinase/phosphatase (PNK). The 5'-OH produced by RegB cleavage is phosphorylated by the kinase activity of PNK. This modification allows host RNases G and E, with activity that is strongly stimulated by 5'-monophosphate termini, to attack mRNAs from the 5'-end, causing their destabilization. The PNK-dependent pathway of degradation becomes effective 5 min postinfection, consistent with our finding that several minutes are required for PNK to accumulate after infection. Our work emphasizes the importance of the nature of the 5' terminus for mRNA stability and depicts a pathway of mRNA degradation with 5'- to 3'-polarity in cells devoid of 5'-3' exonucleases. It also ascribes a role for T4 PNK during normal phage development.

Upregulation of RNase E activity by mutation of a site that uncompetitively interferes with RNA binding

Escherichia coli RNase E contains a site that selectively binds to RNAs containing 5′-monophosphate termini, increasing the efficiency of endonucleolytic cleavage of these RNAs. Random mutagenesis of N-Rne, the N-terminal catalytic region of RNase E, identified a hyperactive variant that remains preferentially responsive to phosphorylation at 5’ termini. Biochemical analyses showed that the mutation (Q36R), which replaces glutamine with arginine at a position distant from the catalytic site, increases formation of stable RNA-protein complexes without detectably affecting the enzyme’s secondary or tertiary structure. Studies of cleavage of fluorogenic substrate and EMSA experiments indicated that the Q36R mutation increases catalytic activity and RNA binding. However, UV crosslinking and mass spectrometry studies suggested that the mutant enzyme lacks an RNA binding site present in its wild-type counterpart: two substrate-bound tryptic peptides, 65HGFLPLK71—which includes amino acids previously implicated in substrate binding and catalysis—and 24LYDLDIESPGHEQK37—which includes the Q36 locus—were identified in wild-type enzyme complexes. Only the shorter peptide was observed for complexes containing Q36R. Our results identify a novel RNase E locus that disparately affects the number of substrate binding sites and catalytic activity of the enzyme. We propose a model that may account for these surprising effects.

The Human Mitochondrial Transcriptome

The human mitochondrial genome comprises a distinct genetic system transcribed as precursor polycistronic transcripts that are subsequently cleaved to generate individual mRNAs, tRNAs, and rRNAs. Here, we provide a comprehensive analysis of the human mitochondrial transcriptome across multiple cell lines and tissues. Using directional deep sequencing and parallel analysis of RNA ends, we demonstrate wide variation in mitochondrial transcript abundance and precisely resolve transcript processing and maturation events. We identify previously undescribed transcripts, including small RNAs, and observe the enrichment of several nuclear RNAs in mitochondria. Using high-throughput in vivo DNaseI footprinting, we establish the global profile of DNA-binding protein occupancy across the mitochondrial genome at single-nucleotide resolution, revealing regulatory features at mitochondrial transcription initiation sites and functional insights into disease-associated variants. This integrated analysis of the mitochondrial transcriptome reveals unexpected complexity in the regulation, expression, and processing of mitochondrial RNA and provides a resource for future studies of mitochondrial function (accessed at http://mitochondria.matticklab.com).

RtcB Is the RNA Ligase Component of an Escherichia coli RNA Repair Operon

RNA 2',3'-cyclic phosphate ends play important roles in RNA metabolism as substrates for RNA ligases during tRNA restriction-repair and tRNA splicing. Diverse bacteria from multiple phyla encode a two-component RNA repair cassette, comprising Pnkp (polynucleotide kinase-phosphatase-ligase) and Hen1 (RNA 3'-terminal ribose 2'-O-methyltransferase), that heals and then seals broken tRNAs with 2',3'-cyclic phosphate and 5'-OH ends. The Pnkp-Hen1 repair operon is absent in the majority of bacterial species, thereby raising the prospect that other RNA repair systems might be extant. A candidate component is RNA 3'-phosphate cyclase, a widely distributed enzyme that transforms RNA 3'-monophosphate termini into 2',3'-cyclic phosphates but cannot seal the ends it produces. Escherichia coli RNA cyclase (RtcA) is encoded in a σ(54)-regulated operon with RtcB, a protein of unknown function. Taking a cue from Pnkp-Hen1, we purified E. coli RtcB and tested it for RNA ligase activity. We report that RtcB per se seals broken tRNA-like stem-loop structures with 2',3'-cyclic phosphate and 5'-OH ends to form a splice junction with a 2'-OH, 3',5'-phosphodiester. We speculate that: (i) RtcB might afford bacteria a means to recover from stress-induced RNA damage; and (ii) RtcB homologs might catalyze tRNA repair or splicing reactions in archaea and eukarya.

Discrimination of phosphorylated double stranded DNA by naphthalene diimide having zinc(II) dipicolylamine complexes

Discrimination of phosphomonoesters and phosphodiesters of DNA was attempted with naphthalene diimide carrying two zinc-dipicolylamine (Dpa) units (1). The binding constant of 1 for a self-complementary octanucleotide was 1.3×10(6)M(-1), while the value for the phosphorylated counterpart was 4.8×10(6)M(-1). This fourfold increase in the binding constant seems to stem from higher affinity of the terminal monophosphate over the phosphodiesters of DNA as the fourth ligand for the metal in 1. Likewise, the binding constant of 1 for DNase I-treated calf thymus DNA (average size 200bp) was twice as large as that for untreated DNA (1kb), possibly because the terminal phosphate groups are five times abundant in the former. These findings provide a clue to developing a system where phosphomonoesters generated upon DNA nicking are discriminated specifically from intact phosphodiesters.

Angiogenin-induced tRNA-derived Stress-induced RNAs Promote Stress-induced Stress Granule Assembly

Angiogenin (ANG) is a secreted ribonuclease that cleaves tRNA to initiate a stress-response program in mammalian cells. Here we show that ANG inhibits protein synthesis and promotes arsenite- and pateamine A-induced assembly of stress granules (SGs). These effects are abrogated in cells transfected with the ANG inhibitor RNH1. Transfection of natural or synthetic 5'- but not 3'-tRNA fragments (tRNA-derived stress-induced RNAs; tiRNAs) induces the phospho-eukaryotic translation initiation factor 2alpha-independent assembly of SGs. Natural 5'-tiRNAs but not 3'-tiRNAs are capped with a 5'-monophosphate that is required for optimal SG assembly. These findings reveal that SG assembly is a component of the ANG- and tiRNA-induced stress response program.

Poly(rC) Binding Proteins and the 5′ Cloverleaf of Uncapped Poliovirus mRNA Function during De Novo Assembly of Polysomes

Poliovirus (PV) mRNA is unusual because it possesses a 5'-terminal monophosphate rather than a 5'-terminal cap. Uncapped mRNAs are typically degraded by the 5' exonuclease XRN1. A 5'-terminal cloverleaf RNA structure interacts with poly(rC) binding proteins (PCBPs) to protect uncapped PV mRNA from 5' exonuclease (K. E. Murray, A. W. Roberts, and D. J. Barton, RNA 7:1126-1141, 2001). In this study, we examined de novo polysome formation using HeLa cell-free translation-replication reactions. PV mRNA formed polysomes coordinate with the time needed for ribosomes to traverse the viral open reading frame (ORF). Nascent PV polypeptides cofractionated with viral polysomes, while mature PV proteins were released from the polysomes. Alterations in the size of the PV ORF correlated with alterations in the size of polysomes with ribosomes present every 250 to 500 nucleotides of the ORF. Eukaryotic initiation factor 4GI (eIF4GI) was cleaved rapidly as viral polysomes assembled and the COOH-terminal portion of eIF4GI cofractionated with viral polysomes. Poly(A) binding protein, along with PCBP 1 and 2, also cofractionated with viral polysomes. A C24A mutation that inhibits PCBP-5'-terminal cloverleaf RNA interactions inhibited the formation and stability of nascent PV polysomes. Kinetic analyses indicated that the PCBP-5' cloverleaf RNA interaction was necessary to protect PV mRNA from 5' exonuclease immediately as ribosomes initially traversed the viral ORF, before viral proteins could alter translation factors within nascent polysomes or contribute to ribonucleoprotein complexes at the termini of the viral mRNA.

Ce(IV)-Catalyzed Site-Selective DNA Hydrolysis Using Tris- and Tetrakismethylenephosphonate-Modified Oligonucleotides

To improve the recruitment of the catalytically active Ce(IV) ion to the DNA cleavage site in the ARCUT (Artificial Restriction DNA Cutter) system, two phosphonate ligands, nitrilotris(methylenephosphonic acid) (NTP) and N,N,N',N'-ethylenediaminetetra-(methylenephosphonic acid) (EDTP), have been linked to the 5'- or 3'-end of oligonucleotides complementary to the target DNA. Compared to their counterparts bearing a terminal monophosphate, these new modified oligonucleotides exhibit a modest to significant increase in the cleavage activity, depending on the target.

Site-selective DNA hydrolysis by combining Ce(IV)/EDTA with monophosphate-bearing oligonucleotides and enzymatic ligation of the scission fragments.

By using two oligonucleotide additives that bear a monophosphate group at the termini through various linkers, gap structures were formed at predetermined positions in substrate DNA, and the monophosphate groups were placed at both edges of these gaps. At pH 7.0 and 37 degrees C, the phosphodiester linkages in the gap sites were efficiently and selectively hydrolyzed by Ce(IV)/EDTA complex (EDTA = ethylenediamine-N,N,N',N'-tetraacetate). The linkages in the middle of the gaps were predominantly hydrolyzed. Compared with DNA scission using oligonucleotide additives that bear no terminal monophosphate, the present scission was much faster (22-fold for a 3-base gap and 14-fold for a 5-base gap) and more site selective. Introduction of one monophosphate group to either edge of the gaps was also effective for promotion of both site selectivity and scission rate. The monophosphate group(s) at the gap site recruits the Ce(IV) to the target site and magnifies the difference in intrinsic reactivity between the target site and the others. Even at higher reaction temperatures, the site selectivity remained satisfactorily high. Furthermore, the fragments formed by the site-selective scission were connected with various oligonucleotides by using DNA ligase, producing desired recombinant DNAs.

Monophosphate as Eminent Ligand to Bind Ce(IV)/EDTA Complex for Site-selective DNA Hydrolysis

Abstract By the use of oligonucleotides bearing a terminal monophosphate, 10-base gap was formed in substrate DNA and the monophosphates were placed at the edge of gap. Upon the treatment of these systems with Ce(IV)/EDTA complex, the gap-site was selectively hydrolyzed. The monophosphates remarkably promoted both the selectivity and the efficiency of scission.

Exonucleolytic degradation of double-stranded RNA by an activity in Xenopus laevis germinal vesicles.

We have identified, in extracts from Xenopus laevis germinal vesicles, a 5' exonuclease activity that cleaves double-stranded RNA (dsRNA). Features of the 5' ends of dsRNAs determine whether the strands are symmetrically or asymmetrically degraded. The activity hydrolyzes in the 5' to 3' direction, releasing 5'-mononucleotides processively, favoring strands with 5'-monophosphate termini; molecules with capped ends are resistant to digestion. Because of its ability to processively digest dsRNA to mononucleotides, we have named the exonuclease Chipper, which could cooperate or compete with Dicer (an endonuclease that produces molecules with a 5'-phosphate) in the processing of dsRNA.

Hydrolysis of phosphomonoesters in nucleotides by cerium(IV) ions. Highly selective hydrolysis of monoester over diester in concentrated buffers

Phosphomonoesters in nucleotides are efficiently hydrolysed by CeIV ions under physiological conditions. The half-lives of the residues at pH 7.2 and 50 °C ([CeIV] = 10 mM) are around 10 min. Phosphomonoester hydrolysis by CeIV ions is faster than the hydrolysis of phosphodiesters. Significantly, the selectivity for monoester hydrolysis over diester hydrolysis is remarkably increased by using concentrated buffer solutions (TRIS and HEPES). In 500 mM TRIS buffer, pdA and dAp are hydrolysed 500- and 580-fold faster than is d(ApA), whereas the corresponding ratios in 50 mM TRIS buffer are 85 and 90 respectively. Selective removal of the terminal monophosphate from d(pApA) is achieved by CeIV in these concentrated buffers.

Acceleration of the Zn2+-promoted phosphodiester hydrolysis of oligonucleotides by the 3′-terminal monophosphate group: intrastrand participation over several nucleoside units

The Zn2+-promoted hydrolysis of the 5′-terminal ribonucleoside phosphodiester bond in chimeric ribo/deoxyribo oligonucleotide 3′-monophosphates, Up(Tp)4 and Up(Tp)9, and their dephosphorylated analogue, Up(Tp)3T, has been studied at various metal ion and substrate concentrations, and in the presence and absence of deoxyribooligonucleotide 3′-monophosphates, (Tp)n, containing no cleavable ribonucleoside phosphodiester bond. The results strongly suggest that the rate-accelerating effect of the 3′-terminal monophosphate group on the phosphodiester hydrolysis is of intramolecular origin: the Zn2+ ion bridges the favoured site of coordination, i.e. the terminal monophosphate group, and the cleaving phosphodiester bond. The 3′-monophosphate group also causes the reaction order in [Zn2 +] to deviate from unity, the values obtained with Up(Tp)3T, Up(Tp)4 and Up(Tp)9 being 1.1, 1.4 and 1.7, respectively. Possibly, the intramolecular participation of the 3′-monophosphate bound Zn2+ ion is facilitated by another Zn2+ ion that stabilizes the folded conformation of the oligonucleotide chain in the reactive Zn2 +/substrate macrochelate.

The effect of the 3′-terminal monophosphate group on the metal-ion-promoted hydrolysis of the phosphodiester bonds of short oligonucleotides

Download options Please wait... Article information DOI https://doi.org/10.1039/P29950001197 Article type Paper Download Citation J. Chem. Soc., Perkin Trans. 2, 1995, 1197-1202 BibTex EndNote MEDLINE ProCite ReferenceManager RefWorks RIS Permissions Request permissions The effect of the 3′-terminal monophosphate group on the metal-ion-promoted hydrolysis of the phosphodiester bonds of short oligonucleotides S. Kuusela, A. Azhayev, A. Guzaev and H. Lönnberg, J. Chem. Soc., Perkin Trans. 2, 1995, 1197 DOI: 10.1039/P29950001197 To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page. If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given. If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page. Read more about how to correctly acknowledge RSC content. Search articles by author Satu Kuusela Alex Azhayev Andrei Guzaev Harri Lönnberg

Histidine tRNA from chicken mitochondria has an uncoded 5'-terminal guanylate residue.

In an attempt to identify the transcription initiation sites in chicken mitochondrial DNA, RNAs capped in vitro using vaccinia guanylyl transferase and [alpha-32P] GTP were analyzed. The most abundant labeled transcript was identified by RNA sequencing as the mitochondrial tRNA(His). Sequence analysis also revealed that this tRNA contains an extra guanylate residue at its 5' end, characteristic of the histidine tRNA family. The respective genomic region was also cloned and sequenced. In contrast to bacteria and the mitochondria of fungi and plants, the extra G of chicken mitochondrial tRNA(His) is not encoded in the gene. Therefore, the guanylate residue must be added post-transcriptionally, as demonstrated for the nuclear tRNA(His) in yeast and Drosophila. Analysis of a capped tRNA(His) precursor of chicken mitochondria suggests that addition of the extra G occurs independently of 3' end maturation. Since in the chicken mitochondrial tRNA(His) the extra G can be efficiently labeled by the capping assay, it should possess a 5'-terminal di- or triphosphate, which contrasts to the 5'-terminal monophosphate proposed for the nuclear encoded tRNA(His). Our results imply that the ability of a mitochondrial RNA to be capped in vitro does not necessarily prove that it contains a transcription initiation site.

Purification and properties of an endonuclease endogenous to rat-liver nuclei

An endonuclease endogenous to rat-liver nuclei has been purified by a series of chromatographic procedures and finally by isoelectric focusing (IEF) electrophoresis. The nuclease fraction prepared by the IEF electrophoresis (IEF fraction) showed a p I value of 5.7 and migrated as a single band to a molecular weight position of 46 000 on an SDS-polyacrylamide gel. The activity for single-stranded DNA was enhanced by 10 mM MgCl 2 and/or by 5–15 mM MgCl 2 in the presence of 2 mM CaCl 2 (an optimum pH, 7.0), but was lowered by CaCl 2 alone and inhibited strongly by ZnCl 2 or MnCl 2. The activity for duplex DNA was rather low, although an optimum condition was 10 mM MgCl 2. In fact, even under this condition, the activity was about 40% lower than that for single-stranded DNA. Moreover, the IEF fraction formed single-strand nicks much more rapidly than double-strand cuts in pBR322 DNA, and preferentially produced deoxyadenosine 5′-monophosphate termini in the DNA. In addition, RNAase activity was also detected in this fraction.