Human RNA 5'-kinase (hClp1) can function as a tRNA splicing enzyme in vivo

Yeast and human Clp1 proteins are homologous components of the mRNA 3′-cleavage-polyadenylation machinery. Recent studies highlighting an association of human Clp1 (hClp1) with tRNA splicing endonuclease and an intrinsic RNA-specific 5′-OH polynucleotide kinase activity of hClp1 have prompted speculation that Clp1 might play a catalytic role in tRNA splicing in animal cells. Here, we show that expression of hClp1 in budding yeast can complement conditional and lethal mutations in the essential 5′-OH RNA kinase module of yeast or plant tRNA ligases. The tRNA splicing activity of hClp1 in yeast is abolished by mutations in the kinase active site. In contrast, overexpression of yeast Clp1 (yClp1) cannot rescue kinase-defective tRNA ligase mutants, and, unlike hClp1, the purified recombinant yClp1 protein has no detectable RNA kinase activity in vitro. Mutations of the yClp1 ATP-binding site do not affect yeast viability. These findings, and the fact that hClp1 cannot complement growth of a yeast clp1Δ strain, indicate that yeast and human Clp1 proteins are not functional orthologs, despite their structural similarity. Although hClp1 can perform the 5′-end-healing step of a yeast-type tRNA splicing pathway in vivo, it is uncertain whether its kinase activity is necessary for tRNA splicing in human cells, given that other mammalian counterparts of yeast-type tRNA repair enzymes are nonessential in vivo.     

Distinctive kinetics and substrate specificities of plant and fungal tRNA ligases

Plant and fungal tRNA ligases are trifunctional enzymes that repair RNA breaks with 2′,3′-cyclic-PO₄ and 5′-OH ends. They are composed of cyclic phosphodiesterase (CPDase) and polynucleotide kinase domains that heal the broken ends to generate the 3′-OH, 2′-PO₄, and 5′-PO₄ required for sealing by a ligase domain. Here, we use short _HORNA>p substrates to determine, in a one-pot assay format under single-turnover conditions, the order and rates of the CPDase, kinase and ligase steps. The observed reaction sequence for the plant tRNA ligase AtRNL, independent of RNA length, is that the CPDase engages first, converting _HORNA>p to _HORNA_2′p, which is then phosphorylated to pRNA_2′p by the kinase. Whereas the rates of the AtRNL CPDase and kinase reactions are insensitive to RNA length, the rate of the ligase reaction is slowed by a factor of 16 in the transition from 10-mer RNA to 8-mer and further by eightfold in the transition from 8-mer RNA to 6-mer. We report that a single ribonucleoside-2′,3′-cyclic-PO₄ moiety enables AtRNL to efficiently splice an otherwise all-DNA strand. Our characterization of a fungal tRNA ligase (KlaTrl1) highlights important functional distinctions vis à vis the plant homolog. We find that (1) the KlaTrl1 kinase is 300-fold faster than the AtRNL kinase; and (2) the KlaTrl1 kinase is highly specific for GTP or dGTP as the phosphate donor. Our findings recommend tRNA ligase as a tool to map ribonucleotides embedded in DNA and as a target for antifungal drug discovery.     

RtcB, a novel RNA ligase, can catalyze tRNA splicing and HAC1 mRNA splicing in vivo

RtcB enzymes are novel RNA ligases that join 2',3'-cyclic phosphate and 5'-OH ends. The phylogenetic distribution of RtcB points to its candidacy as a tRNA splicing/repair enzyme. Here we show that Escherichia coli RtcB is competent and sufficient for tRNA splicing in vivo by virtue of its ability to complement growth of yeast cells that lack the endogenous "healing/sealing-type" tRNA ligase Trl1. RtcB also protects yeast trl1Δ cells against a fungal ribotoxin that incises the anticodon loop of cellular tRNAs. Moreover, RtcB can replace Trl1 as the catalyst of HAC1 mRNA splicing during the unfolded protein response. Thus, RtcB is a bona fide RNA repair enzyme with broad physiological actions. Biochemical analysis of RtcB highlights the uniqueness of its active site and catalytic mechanism. Our findings draw attention to tRNA ligase as a promising drug target.     

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.     

Structure–activity relationships in human RNA 3′-phosphate cyclase

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 Rtc1 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. Here we used the crystal structure of Escherichia coli RtcA to guide a mutational analysis of the human RNA cyclase Rtc1. An alanine scan defined seven conserved residues as essential for the Rtc1 RNA cyclization and autoadenylylation reactions. Structure–activity relationships were clarified by conservative substitutions. Our results are consistent with a mechanism of adenylate transfer in which attack of the Rtc1 His320 nucleophile on the ATP α phosphorus is facilitated by proper orientation of the PP_i leaving group via contacts to Arg21, Arg40, and Arg43. We invoke roles for Tyr294 in binding the adenine base and Glu14 in binding the divalent cation cofactor. We find that Rtc1 forms a stable binary complex with a 3′-phosphate terminated RNA, but not with an otherwise identical 3′-OH terminated RNA. Mutation of His320 had little impact on RNA 3′-phosphate binding, signifying that covalent adenylylation of Rtc1 is not a prerequisite for end recognition.     

The brain in vivo expresses the 2',3'-cAMP-adenosine pathway

Although multiple biochemical pathways produce adenosine, studies suggest that the 2',3'-cAMP-adenosine pathway (2',3'-cAMP→2'-AMP/3'-AMP→adenosine) contributes to adenosine production in some cells/tissues/organs. To determine whether the 2',3'-cAMP-adenosine pathway exists in vivo in the brain, we delivered to the brain (gray matter and white matter separately) via the inflow perfusate of a microdialysis probe either 2',3'-cAMP, 3',5'-cAMP, 2'-AMP, 3'-AMP, or 5'-AMP and measured the recovered metabolites in the microdialysis outflow perfusate with mass spectrometry. In both gray and white matter, 2',3'-cAMP increased 2'-AMP, 3'-AMP and adenosine, and 3',5'-cAMP increased 5'-AMP and adenosine. In both brain regions, 2'-AMP, 3-AMP and 5'-AMP were converted to adenosine. Microdialysis experiments in 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) wild-type mice demonstrated that traumatic brain injury (controlled cortical impact model) activated the brain 2',3'-cAMP-adenosine pathway; similar experiments in CNPase knockout mice indicated that CNPase was involved in the metabolism of endogenous 2',3'-cAMP to 2'-AMP and to adenosine. In CSF from traumatic brain injury patients, 2',3'-cAMP was significantly increased in the initial 12 h after injury and strongly correlated with CSF levels of 2'-AMP, 3'-AMP, adenosine and inosine. We conclude that in vivo, 2',3'-cAMP is converted to 2'-AMP/3'-AMP, and these AMPs are metabolized to adenosine. This pathway exists endogenously in both mice and humans.     

A role for tRNA(His) guanylyltransferase (Thg1)-like proteins from Dictyostelium discoideum in mitochondrial 5'-tRNA editing

Genes with sequence similarity to the yeast tRNA(His) guanylyltransferase (Thg1) gene have been identified in all three domains of life, and Thg1 family enzymes are implicated in diverse processes, ranging from tRNA(His) maturation to 5'-end repair of tRNAs. All of these activities take advantage of the ability of Thg1 family enzymes to catalyze 3'-5' nucleotide addition reactions. Although many Thg1-containing organisms have a single Thg1-related gene, certain eukaryotic microbes possess multiple genes with sequence similarity to Thg1. Here we investigate the activities of four Thg1-like proteins (TLPs) encoded by the genome of the slime mold, Dictyostelium discoideum (a member of the eukaryotic supergroup Amoebozoa). We show that one of the four TLPs is a bona fide Thg1 ortholog, a cytoplasmic G(-1) addition enzyme likely to be responsible for tRNA(His) maturation in D. discoideum. Two other D. discoideum TLPs exhibit biochemical activities consistent with a role for these enzymes in mitochondrial 5'-tRNA editing, based on their ability to efficiently repair the 5' ends of mitochondrial tRNA editing substrates. Although 5'-tRNA editing was discovered nearly two decades ago, the identity of the protein(s) that catalyze this activity has remained elusive. This article provides the first identification of any purified protein that appears to play a role in the 5'-tRNA editing reaction. Moreover, the presence of multiple Thg1 family members in D. discoideum suggests that gene duplication and divergence during evolution has resulted in paralogous proteins that use 3'-5' nucleotide addition reactions for diverse biological functions in the same organism.     

Expression of the 2',3'-cAMP-adenosine pathway in astrocytes and microglia

Many organs express the extracellular 3',5'-cAMP-adenosine pathway (conversion of extracellular 3',5'-cAMP to 5'-AMP and 5'-AMP to adenosine). Some organs release 2',3'-cAMP (isomer of 3',5'-cAMP) and convert extracellular 2',3'-cAMP to 2'- and 3'-AMP and convert these AMPs to adenosine (extracellular 2',3'-cAMP-adenosine pathway). As astrocytes and microglia are important participants in the response to brain injury and adenosine is an endogenous neuroprotectant, we investigated whether these extracellular cAMP-adenosine pathways exist in these cell types. 2',3'-, 3',5'-cAMP, 5'-, 3'-, and 2'-AMP were incubated with mouse primary astrocytes or primary microglia for 1 h and purine metabolites were measured in the medium by mass spectrometry. There was little evidence of a 3',5'-cAMP-adenosine pathway in either astrocytes or microglia. In contrast, both cell types converted 2',3'-cAMP to 2'- and 3'-AMP (with 2'-AMP being the predominant product). Although both cell types converted 2'- and 3'-AMP to adenosine, microglia were five- and sevenfold, respectively, more efficient than astrocytes in this regard. Inhibitor studies indicated that the conversion of 2',3'-cAMP to 2'-AMP was mediated by a different ecto-enzyme than that involved in the metabolism of 2',3'-cAMP to 3'-AMP and that although CD73 mediates the conversion of 5'-AMP to adenosine, an alternative ecto-enzyme metabolizes 2'- or 3'-AMP to adenosine.     

Doing it in reverse: 3'-to-5' polymerization by the Thg1 superfamily

The tRNA(His) guanylyltransferase (Thg1) family of enzymes comprises members from all three domains of life (Eucarya, Bacteria, Archaea). Although the initial activity associated with Thg1 enzymes was a single 3'-to-5' nucleotide addition reaction that specifies tRNA(His) identity in eukaryotes, the discovery of a generalized base pair-dependent 3'-to-5' polymerase reaction greatly expanded the scope of Thg1 family-catalyzed reactions to include tRNA repair and editing activities in bacteria, archaea, and organelles. While the identification of the 3'-to-5' polymerase activity associated with Thg1 enzymes is relatively recent, the roots of this discovery and its likely physiological relevance were described ≈ 30 yr ago. Here we review recent advances toward understanding diverse Thg1 family enzyme functions and mechanisms. We also discuss possible evolutionary origins of Thg1 family-catalyzed 3'-to-5' addition activities and their implications for the currently observed phylogenetic distribution of Thg1-related enzymes in biology.     

Novel one-step mechanism for tRNA 3'-end maturation by the exoribonuclease RNase R of Mycoplasma genitalium

Mycoplasma genitalium is expected to metabolize RNA using unique pathways because its minimal genome encodes very few ribonucleases. In this work, we report that the only exoribonuclease identified in M. genitalium, RNase R, is able to remove tRNA 3'-trailers and generate mature 3'-ends. Several sequence and structural features of a tRNA precursor determine its precise processing at the 3'-end by RNase R in a purified system. The aminoacyl-acceptor stem plays a major role in stopping RNase R digestion at the mature 3'-end. Disruption of the stem causes partial or complete degradation of the pre-tRNA by RNase R, whereas extension of the stem results in the formation of a product terminating downstream at the new mature 3'-end. In addition, the 3'-terminal CCA sequence and the discriminator residue influence the ability of RNase R to stop at the mature 3'-end. RNase R-mediated generation of the mature 3'-end prefers a sequence of RCCN at the 3' terminus of tRNA. Variations of this sequence may cause RNase R to trim further and remove terminal CA residues from the mature 3'-end. Therefore, M. genitalium RNase R can precisely remove the 3'-trailer of a tRNA precursor by recognizing features in the terminal domains of tRNA, a process requiring multiple RNases in most bacteria.     

Inhibition of calmodulin-dependent cyclic nucleotide phosphodiesterase by flunarizine, a calcium-entry blocker

It has been reported that flunarizine, classified as calcium entry-blockers, is a potent brain protective drug without any heart depressant effect, contrasting with other drugs in this group. This paper presents evidence that through a competitive antagonism against calmodulin, a major intracellular calcium receptor, flunarizine inhibits the calcium X calmodulin-activated phosphodiesterase activity of bovine brain, but not of heart, whereas other calcium-entry blockers and calmodulin antagonists inhibit to the same extent, the activation of the enzyme from the two sources. It could be suggested that some of pharmacological effects by flunarizine and its differences from other calcium-entry blockers may be explained by its interaction with calmodulin.     

(2'-5')An-dependent endoribonuclease: enzyme levels are regulated by IFN beta, IFN gamma, and cell culture conditions

The levels of a (2'-5')An-dependent endonuclease (RNase L) were determined in extracts prepared from murine L cells and Ehrlich ascites tumor (EAT) cells by measuring specific binding of protein to a labeled derivative of (2'-5')An, (2'-5')A3[32P]pCp. RNase L levels were found to depend both on interferon (IFN) treatment and on cell growth conditions. Treatment of murine L cells and EAT cells with 100-2,000 IRU IFN beta or IFN gamma resulted in a similar 2-4-fold increase in the levels of RNase L when cells were present at low density. The levels of RNase L were also shown to increase 2-3-fold as cells approached saturation density. Serum-starved cells also displayed relatively high levels of RNase L. RNase L levels in cells maintained at high cell density did not change appreciably following treatment with IFN beta or IFN gamma. Regulation of RNase L levels by cell growth conditions as well as by IFN beta or IFN gamma treatment suggests that RNase L may play an important role in regulating the levels of cellular mRNAs as well as acting to degrade viral RNAs.     

Molecular architecture of a miRNA-regulated 3' UTR

Animal genomes contain hundreds of microRNAs (miRNAs), small regulatory RNAs that control gene expression by binding to complementary sites in target mRNAs. Some rules that govern miRNA/target interaction have been elucidated but their general applicability awaits further experimentation on a case-by-case basis. We use here an assay system in transgenic nematodes to analyze the interaction of the Caenorhabditis elegans lsy-6 miRNA with 3' UTR sequences. In contrast to many previously described assay systems used to analyze miRNA/target interactions, our assay system operates within the cellular context in which lsy-6 normally functions, a single neuron in the nervous system of C. elegans. Through extensive mutational analysis, we define features in the known and experimentally validated target of lsy-6, the 3' UTR of the cog-1 homeobox gene, that are required for a functional miRNA/target interaction. We describe that both in the context of the cog-1 3' UTR and in the context of heterologous 3' UTRs, one or more seed matches are not a reliable predictor for a functional miRNA/target interaction. We rather find that two nonsequence specific contextual features beyond miRNA target sites are critical determinants of miRNA-mediated 3' UTR regulation. The contextual features reside 3' of lsy-6 binding sites in the 3' UTR and act in a combinatorial manner; mutation of each results in limited defects in 3' UTR regulation, but a combinatorial deletion results in complete loss of 3' UTR regulation. Together with two lsy-6 sites, these two contextual features are capable of imparting regulation on a heterologous 3' UTR. Moreover, the contextual features need to be present in a specific configuration relative to miRNA binding sites and could either represent protein binding sites or provide an appropriate structural context. We conclude that a given target site resides in a 3' UTR context that evolved beyond target site complementarity to support regulation by a specific miRNA. The large number of 3' UTRs that we analyzed in this study will also be useful to computational biologists in designing the next generation of miRNA/target prediction algorithms.     

The highly conserved 5' untranslated region as an effective target towards the inhibition of Enterovirus 71 replication by unmodified and appropriate 2'-modified siRNAs

Background

Cryptococcus neoformans has a predilection for central nervous system infection. C. neoformans traversal of the blood brain barrier, composed of human brain microvascular endothelial cells (HBMEC), is the crucial step in brain infection. However, the molecular mechanism of the interaction between Cryptococcus neoformans and HBMEC, relevant to its brain invasion, is still largely unknown.

Methods

In this report, we explored several cellular and molecular events involving the membrane lipid rafts and caveolin-1 (Cav1) of HBMEC during C. neoformans infection. Immunofluorescence microscopy was used to examine the roles of Cav1. The knockdown of Cav1 by the siRNA treatment was performed. Phosphorylation of Cav1 relevant to its invasion functions was investigated.

Results

We found that the host receptor CD44 colocalized with Cav1 on the plasma membrane, and knockdown of Cav1 significantly reduced the fungal ability to invade HBMEC. Although the CD44 molecules were still present, HBMEC membrane organization was distorted by Cav1 knockdown. Concomitantly, knockdown of Cav1 significantly reduced the fungal crossing of the HBMEC monolayer in vitro. Upon C. neoformans engagement, host Cav1 was phosphorylated in a CD44-dependent manner. This phosphorylation was diminished by filipin, a disrupter of lipid raft structure. Furthermore, the phosphorylated Cav1 at the lipid raft migrated inward to the perinuclear localization. Interestingly, the phospho-Cav1 formed a thread-like structure and colocalized with actin filaments but not with the microtubule network.

Conclusion

These data support that C. neoformans internalization into HBMEC is a lipid raft/caveolae-dependent endocytic process where the actin cytoskeleton is involved, and the Cav1 plays an essential role in C. neoformans traversal of the blood-brain barrier.     

1-[2-Hydroxy-3-octadecan-1'-oate]propyl-2'',2'',5'',5''-tetramethyl pyrolidine-N-oxyl-3''-carboxylate as a potential spin probe for membrane structure studies

The synthesis of a new minimum steric perturbing proxyl nitroxide, which is a derivative of glycerol and contains a stearic acid moiety, has been carried out. Its localization in model membrane L-alpha-dipalmitoyl phosphatidyl choline (DPPC) was ascertained with the help of ESR, DSC, 1H and 31P NMR techniques. The nitroxide was used for detecting the changes in the phase transition temperature of the model membranes in the presence and absence of drugs. The permeation of the vasodilating drug epinephrine has also been studied using this spin label. The results prove the potential applicability of the new spin probe in the spin labeling of biomembranes.     

Characterization of (2S,2'R,3'R)-2-(2',3'-[3H]-Dicarboxycyclopropyl)glycine Binding in Rat Brain

Abstract: [(2S,2′R,3′R)-2-(2′,3′-[³H]Dicarboxycyclopropyl)glycine ([³H]DCG IV) binding was characterized in vitro in rat brain cortex homogenates and rat brain sections. In cortex homogenates, the binding was saturable and the saturation isotherm indicated the presence of a single binding site with a K_D value of 180 ± 33 nM and a B_max of 780 ± 70 fmol/mg of protein. The nonspecific binding, measured using 100 µM LY354740, was <30%. NMDA, AMPA, kainate, l (?)-threo-3-hydroxyaspartic acid, and (S)-3,5-dihydroxyphenylglycine were all inactive in [³H]DCG IV binding up to 1 mM. However, several compounds inhibited [³H]DCG IV binding in a concentration-dependent manner with the following rank order of potency: LY341495 = LY354740 > DCG IV = (2S,1′S,2′S)-2-(2-carboxycyclopropyl)glycine > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid > (2S,1′S,2′S)-2-methyl-2-(2-carboxycyclopropyl)glycine > l -glutamate = ibotenate > quisqualate > (RS)-α-methyl-4-phosphonophenylglycine = l (+)-2-amino-3-phosphonopropionic acid > (S)-α-methyl-4-carboxyphenylglycine > (2S)-α-ethylglutamic acid > l (+)-2-amino-4-phosphonobutyric acid. N-Acetyl-l -aspartyl-l -glutamic acid inhibited the binding in a biphasic manner with an IC₅₀ of 0.2 µM for the high-affinity component. The binding was also affected by GTPγS, reducing agents, and CdCl₂. In parasagittal sections of rat brain, a high density of specific binding was observed in the accessory olfactory bulb, cortical regions (layers 1, 3, and 4 > 2, 5, and 6), caudate putamen, molecular layers of the hippocampus and dentate gyrus, subiculum, presubiculum, retrosplenial cortex, anteroventral thalamic nuclei, and cerebellar granular layer, reflecting its preferential (perhaps not exclusive) affinity for pre- and postsynaptic metabotropic glutamate mGlu2 receptors. Thus, the pharmacology, tissue distribution, and sensitivity to GTPγS show that [³H]DCG IV binding is probably to group II metabotropic glutamate receptors in rat brain.