Pathways controlling dNTP pools to maintain genome stability.

Artificially modified nucleotides, in the form of nucleoside analogues, are widely used in the treatment of cancers and various other diseases, and have become important tools in the laboratory to characterise DNA repair pathways. In contrast, the role of endogenously occurring nucleotide modifications in genome stability is little understood. This is despite the demonstration over three decades ago that the cellular DNA precursor pool is orders of magnitude more susceptible to modification than the DNA molecule itself. More recently, underscoring the importance of this topic, oxidation of the cellular nucleotide pool achieved through targeting the sanitation enzyme MTH1, appears to be a promising anti-cancer strategy. This article reviews our current understanding of modified DNA precursors in genome stability, with a particular focus upon oxidised nucleotides, and outlines some important outstanding questions.

Platelet-Derived Growth Factor Receptor-α Regulates Proliferation of Gastrointestinal Stromal Tumor Cells With Mutations in KIT by Stabilizing ETV1.

BACKGROUND & AIMS: In gastrointestinal muscles, v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) is predominantly expressed by interstitial cells of Cajal (ICC) and platelet-derived growth factor receptor-α (PDGFRA) polypeptide is expressed by so-called fibroblast-like cells. KIT and PDGFRA have been reported to be coexpressed in ICC precursors and gastrointestinal stromal tumors (GISTs), which originate from the ICC lineage. PDGFRA signaling has been proposed to stimulate growth of GISTs that express mutant KIT, but the effects and mechanisms of selective blockade of PDGFRA are unclear. We investigated whether inhibiting PDGFRA could reduce proliferation of GIST cells with mutant KIT via effects on the KIT-dependent transcription factor ETV1. METHODS: We studied 53 gastric, small intestinal, rectal, or abdominal GISTs collected immediately after surgery or archived as fixed blocks at the Mayo Clinic and University of California, San Diego. In human GIST cells carrying imatinib-sensitive and imatinib-resistant mutations in KIT, PDGFRA was reduced by RNA interference (knockdown) or inhibited with crenolanib besylate (a selective inhibitor of PDGFRA and PDGFRB). Mouse ICC precursors were retrovirally transduced to overexpress wild-type Kit. Cell proliferation was analyzed by methyltetrazolium, 5-ethynyl-2'-deoxyuridine incorporation, and Ki-67 immunofluorescence assays; we also analyzed growth of xenograft tumors in mice. Gastric ICC and ICC precursors, and their PDGFRA(+) subsets, were analyzed by flow cytometry and immunohistochemistry in wild-type, Kit(+/copGFP), Pdgfra(+/eGFP), and NOD/ShiLtJ mice. Immunoblots were used to quantify protein expression and phosphorylation. RESULTS: KIT and PDGFRA were coexpressed in 3%-5% of mouse ICC, 35%-44% of ICC precursors, and most human GIST samples and cell lines. PDGFRA knockdown or inhibition with crenolanib efficiently reduced proliferation of imatinib-sensitive and imatinib-resistant KIT(+)ETV1(+)PDGFRA(+) GIST cells (50% maximal inhibitory concentration = 5-32 nM), but not of cells lacking KIT, ETV1, or PDGFRA (50% maximal inhibitory concentration >230 nM). Crenolanib inhibited phosphorylation of PDGFRA and PDGFRB, but not KIT. However, Kit overexpression sensitized mouse ICC precursors to crenolanib. ETV1 knockdown reduced KIT expression and GIST proliferation. Crenolanib down-regulated ETV1 by inhibiting extracellular-signal-regulated kinase (ERK)-dependent stabilization of ETV1 protein and also reduced expression of KIT and PDGFRA. CONCLUSIONS: In KIT-mutant GIST, inhibition of PDGFRA disrupts a KIT-ERK-ETV1-KIT signaling loop by inhibiting ERK activation. The PDGFRA inhibitor crenolanib might be used to treat patients with imatinib-resistant, KIT-mutant GIST.

The deoxynucleotide triphosphohydrolase SAMHD1 is a major regulator of DNA precursor pools in mammalian cells.

Sterile alpha motif and HD-domain containing protein 1 (SAMHD1) is a triphosphohydrolase converting deoxynucleoside triphosphates (dNTPs) to deoxynucleosides. The enzyme was recently identified as a component of the human innate immune system that restricts HIV-1 infection by removing dNTPs required for viral DNA synthesis. SAMHD1 has deep evolutionary roots and is ubiquitous in human organs. Here we identify a general function of SAMHD1 in the regulation of dNTP pools in cultured human cells. The protein was nuclear and variably expressed during the cell cycle, maximally during quiescence and minimally during S-phase. Treatment of lung or skin fibroblasts with specific siRNAs resulted in the disappearence of SAMHD1 accompanied by loss of the cell-cycle regulation of dNTP pool sizes and dNTP imbalance. Cells accumulated in G1 phase with oversized pools and stopped growing. Following removal of the siRNA, the pools were normalized and cell growth restarted, but only after SAMHD1 had reappeared. In quiescent cultures SAMHD1 down-regulation leads to a marked expansion of dNTP pools. In all cases the largest effect was on dGTP, the preferred substrate of SAMHD1. Ribonucleotide reductase, responsible for the de novo synthesis of dNTPs, is a cytosolic enzyme maximally induced in S-phase cells. Thus, in mammalian cells the cell cycle regulation of the two main enzymes controlling dNTP pool sizes is adjusted to the requirements of DNA replication. Synthesis by the reductase peaks during S-phase, and catabolism by SAMHD1 is maximal during G1 phase when large dNTP pools would prevent cells from preparing for a new round of DNA replication.

Analyzing modular RNA structure reveals low global structural entropy in microRNA sequence.

Secondary structure remains the most exploitable feature for noncoding RNA (ncRNA) gene finding in genomes. However, methods based on secondary structure prediction may generate superfluous amount of candidates for validation and have yet to deliver the desired performance that can complement experimental efforts in ncRNA gene finding. This paper investigates a novel method, unpaired structural entropy (USE) as a measurement for the structure fold stability of ncRNAs. USE proves to be effective in identifying from the genome background a class of ncRNAs, such as precursor microRNAs (pre-miRNAs) that contains a long stem hairpin loop. USE correlates well and performs better than other measures on pre-miRNAs, including the previously formulated structural entropy. As an SVM classifier, USE outperforms existing pre-miRNA classifiers. A long stem hairpin loop is common for a number of other functional RNAs including introns splicing hairpins loops and intrinsic termination hairpin loops. We believe USE can be further applied in developing ab initio prediction programs for a larger class of ncRNAs.

Comparative genomics reveals novel biochemical pathways.

How well do we understand which enzymes are involved in the primary metabolism of the cell? A recent study using comparative genomics and postgenomics approaches revealed a novel pathway in the most studied organism, Escherichia coli. The analysis of a new operon consisting of seven previously uncharacterized genes thought to be involved in the degradation of nucleic acid precursors shows the impact of comparative genomics on the discovery of novel pathways and enzymes.

DNA precursor asymmetries in mammalian tissue mitochondria and possible contribution to mutagenesis through reduced replication fidelity.

The mutation rate of the mammalian mitochondrial genome is higher than that of the nuclear genome. Because mitochondrial and nuclear deoxyribonucleoside triphosphate (dNTP) pools are physically distinct and because dNTP concentrations influence replication fidelity, we asked whether mitochondrial dNTP pools are asymmetric with respect to each other. We report here that the concentrations of the four dNTPs are not equal in mitochondria isolated from several tissues of both young and old rats. In particular, in most tissues examined, mitochondrial dGTP concentrations are high relative to the other dNTPs. Moreover, in the presence of the biased dNTP concentrations measured in heart and skeletal muscle, the fidelity of DNA synthesis in vitro by normally highly accurate mtDNA polymerase gamma is reduced, with error frequencies increased by as much as 3-fold, due to increased formation of template T.dGTP mismatches that are inefficiently corrected by proofreading. These data, plus some published data on specific mitochondrial mutations seen in human diseases, are consistent with the hypothesis that normal intramitochondrial dNTP pool asymmetries may contribute to spontaneous mutagenesis in the mammalian mitochondrial genome.

Functional interaction between RNase III and the Escherichia coli ribosome.

BACKGROUND: RNase III is a dsRNA specific endoribonuclease which is involved in the primary processing of rRNA and several mRNA species in bacteria. Both primary structural elements and the secondary structure of the substrate RNA play a role in cleavage specificity. RESULTS: We have analyzed RNase III cleavage sites around both ends of pre-23 S rRNA in the ribosome and in the protein-free pre-rRNA. It was found that in the protein-free pre-23 S rRNA the main cleavage site is at position (-7) in respect of the mature 5' end. When pre-23 S rRNA was in 70 S ribosomes or in 50 S subunits, the RNase III cleavage occurred at position (-3). We have demonstrated that RNase III interacts with both ribosomal subunits and with even higher affinity with 70 S ribosomes. Association of RNase III with 70 S ribosomes cannot be dissociated by poly(U) RNA indicating that the binding is specific. CONCLUSIONS: In addition to the primary and secondary structural elements in RNA, protein binding to substrate RNA can be a determinant of the RNase III cleavage site.

Uncoupling yeast intron recognition from transcription with recursive splicing.

Pre-mRNA splicing has to be coordinated with other processes occurring in the nucleus including transcription, mRNA 3' end formation and mRNA export. To analyze the relationship between transcription and splicing, we constructed a network of nested introns. Introns were inserted in the 5' splice site and/or branchpoint of a synthetic yeast intron interrupting a reporter gene. The inserted introns mask the recipient intron from the cellular machinery until they are removed by splicing. Production of functional mRNA from these constructs therefore requires recognition of a spliced RNA as a splicing substrate. We show that recurrent splicing occurs in a sequential and ordered fashion in vivo. Thus, in Saccharomyces cerevisiae, intron recognition and pre-spliceosome assembly is not tightly coupled to transcription.

The effect of long-range loop-loop interactions on folding of the Tetrahymena self-splicing RNA.

Bas?e-pairing between the terminal loops of helices P2.1 and P9.1a (P13) and P2 and P5c (P14) stabilize the folded structure of the Tetrahymena group I intron. Using native gel electrophoresis to analyze the folding kinetics of a natural pre-RNA containing the Tetrahymena intron, we show that P13 and P14 are the only native loop-loop interactions among six possible combinations. Other base-pairing interactions of the loop sequences stabilize misfolded and inactive pre-RNAs. Mismatches in P13 or P14 raised the midpoints and decreased the cooperativity of the Mg(2+)-dependent eqXuilibrium folding transitions. Although some mutations in P13 resulted in slightly higher folding rates, others led to slower folding compared to the wild-type, suggesting that P13 promotes formation of P3 and P7. In contrast, mismatches in P14 increased the rate of folding, suggesting that base-pairing between P5c and P2 stabilizes intermediates in which the catalytic core is misfolded. Although the peripheral helices stabilize the native structure of the catalytic core, our results show that formation of long-range interactions, and competition between correct and incorrect loop-loop base-pairs, decrease the rate at which the active pre-RNA structure is assembled.

Yeast RNase III as a key processing enzyme in small nucleolar RNAs metabolism.

The variety of biogenesis pathways for small nucleolar RNAs (snoRNAs) reflects the diversity of their genomic organization. We have searched for yeast snoRNAs which are affected by the depletion of the yeast ortholog of bacterial RNase III, Rnt1. In a yeast strain inactivated for RNT1, almost half of the snoRNAs tested are depleted with significant accumulation of monocistronic or polycistronic precursors. snoRNAs from both major families of snoRNAs (C/D and H/ACA) are affected by RNT1 disruption. In vitro, recombinant Rnt1 specifically cleaves pre-snoRNA precursors in the absence of other factors, generating intermediates which require the action of other enzymes for processing to the mature snoRNA. Most Rnt1 cleavage sites fall within potentially double-stranded regions closed by tetraloops with a novel consensus sequence AGNN. These results demonstrate that biogenesis of a large number of snoRNAs from the two major families of snoRNAs requires a common RNA endonuclease and a putative conserved structural motif.

[Analysis of polymorphisms Sma (Hpa II) and Sca I gene precursors of atrial natriuretic peptide (ANP) in patients with essential hypertension]. / Analiza polimorfizmów Sma (Hpa II) i Sca I genu prekursora przedsionkowego peptydu natriuretycznego (ANP) u chorych z samoistnym nadcisnieniem tetniczym.

Both environmental and genetic factors are implicated in the pathogenesis of essential hypertension. The defect of the ANP precursor gene leading to the decrease of ANP synthesis are a cause of the development of sodium-sensitive hypertension in animals. Recent findings in African-Americans who are a model of sodium-sensitive population, reveal a strong association between Sma I polymorphism at intron 2 (the polymorphic site is identical for Hpa II restriction enzyme) or both Sma I and Sca I polymorphism at exon 3 of ANP precursor gene and essential hypertension. The aim of our study was to optimize the methods for Sma I and Sca I analysis in the ANP precursor gene (PCR followed by digestion with restriction enzymes) and to determine the frequencies of Sma I or Sca I genotypes and alleles in patients with sodium-sensitive (SS) or sodium-nonsensitive (SR) hypertension. The Sma I heterozygous mutation (WM genotype) were detected in 4 (8.9%) SS patients and in 2 (10%) patients in SR group. The frequency of Sca I M allele (allele with mutation) was significantly higher in SS group as compared to sodium-nonsensitive hypertensives. Our results suggest that, in contrast to Black hypertensives, in Caucasians with essential hypertension the Sma I polymorphism is very rare and the Sca I polymorphism of ANP precursor gene is associated with sodium-sensitivity of blood pressure.

HIV genetic variation is directed and restricted by DNA precursor availability.

The effects of deoxynucleoside triphosphate (dNTP) imbalances on the fidelity of human immunodeficiency virus type 1 (HIV-1) replication were investigated. Using detergent permeabilized virions and biased dNTP concentrations different types of hypermutants were readily produced. However, the mutant spectrum was different from naturally occurring hypermutants demonstrating that the host cell may restrict variation. Using a genetic screen based on the blue/white beta-galactosidase complementation assay, G --> A hypermutants were recovered from HIV-infected thymidine treated U937 cells. Furthermore, hypermutants were recovered from 1 to 2% of resting or activated peripheral blood mononuclear cells indicating that small proportions of primary cells had distorted intracellular [dTTP] and [dCTP]. Such imbalances may underlie a proportion of somatic and germline point mutations and shape to some extent the evolution of mammalian and viral genomes.

In vivo mobility of a group I twintron in nuclear ribosomal DNA of the myxomycete Didymium iridis.

DiSSU1 is an optional group I twintron present in the nuclear extrachromosomal ribosomal DNA of the myxomycete Didymium iridis. DiSSU1 appears to be complex both in structure and function. At the RNA level it has a twin-ribozyme organization composed of two group I ribozymes with different functions, separated by an open reading frame. Here, we show that DiSSU1 is mobile when haploid intron-containing and intron-less amoebae are mated. The mobility process is fast, being completed in 5-10 nuclear cycles after mating in the developing zygote and plasmodia. Analyses of progeny from genetic crosses confirm intron mobility. DiSSU1 is the first example of a mobile group I twintron. The intron-encoded protein was expressed in Escherichia coli and found to be an endonuclease, I-DirI, that cleaves an intron-less ribosomal DNA allele at the intron-insertion site, and is probably involved in intron homing. The endonuclease I-DirI seems to be a rare example of a protein that is expressed from a ribozyme-processed RNA polymerase I transcript in vivo.

An mRNA-type intron is present in the Rhodotorula hasegawae U2 small nuclear RNA gene.

Splicing an mRNA precursor requires multiple factors involving five small nuclear RNA (snRNA) species called U1, U2, U4, U5, and U6. The presence of mRNA-type introns in the U6 snRNA genes of some yeasts led to the hypothesis that U6 snRNA may play a catalytic role in pre-mRNA splicing and that the U6 introns occurred through reverse splicing of an intron from an mRNA precursor into a catalytic site of U6 snRNA. We characterized the U2 snRNA gene of the yeast Rhodotorula hasegawae, which has four mRNA-type introns in the U6 snRNA gene, and found an mRNA-type intron of 60 bp. The intron of the U2 snRNA gene is present in the highly conserved region immediately downstream of the branch site recognition domain. Interestingly, we found that this region can form a novel base pairing with U6 snRNA. We discuss the possible implications of these findings for the mechanisms of intron acquisition and for the role of U2 snRNA in pre-mRNA splicing.

Species-specific differential cleavage and polyadenylation of plasminogen activator inhibitor type 1 hnRNA.

Plasminogen activator inhibitor type 1 (PAI-1) is the primary physiologic inhibitor of the naturally occurring plasminogen activators. In higher primates two forms of mature PAI-1 mRNA (3.2 kb and 2.2 kb) arise by alternative cleavage and polyadenylation of PAI-1 hnRNA which is regulated in a tissue-specific fashion in humans. In other mammals only the 3.2 kb mRNA has been detected. The putative downstream polyadenylation site in humans that gives rise to the 3.2 kb PAI-1 mRNA consists of three overlapping copies of the consensus polyadenylation sequence while no consensus polyadenylation sequence is found upstream at a position that could generate the shorter mRNA species. To determine whether differential cleavage and polyadenylation of PAI-1 mRNA is due to species-specific differences in trans-acting factors that process PAI-1 mRNA or to the presence of a nonconsensus polyadenylation site acquired recently during primate evolution we prepared plasmids in which the 3' nontranslated region of the human PAI-1 gene or the mouse PAI-1 cDNA was inserted downstream of the neomycin gene in the plasmid pSV2neo. We show that the 3'-nontranslated region of the human PAI-1 gene but not the mouse PAI-1 cDNA conferred alternative cleavage and polyadenylation to the neomycin gene in transfected human Hep G2 cells as well as mouse NIH3T3 and rat L6 cells.

RNA B is the major nucleolar trimethylguanosine-capped small nuclear RNA associated with fibrillarin and pre-rRNAs in Trypanosoma brucei.

RNA B is one of three abundant trimethylguanosine-capped U small nuclear RNAs (snRNAs) of Trypanosoma brucei which is not strongly identified with other U snRNAs by sequence homology. We show here that RNA B is a highly diverged U3 snRNA homolog likely involved in pre-rRNA processing. Sequence identity between RNA B and U3 snRNAs is limited; only two of four boxes of homology conserved between U3 snRNAs are obvious in RNA B. These are the box A homology, specific for U3 snRNAs, and the box C homology, common to nucleolar snRNAs and required for association with the nucleolar protein, fibrillarin. A 35-kDa T. brucei fibrillarin homolog was identified by using an anti-Physarum fibrillarin monoclonal antibody. RNA B and fibrillarin were localized in nucleolar fractions of the nucleus which contained pre-rRNAs and did not contain nucleoplasmic snRNAs. Fibrillarin and RNA B were precipitated by scleroderma patient serum S4, which reacts with fibrillarins from diverse organisms; RNA B was the only trimethylguanosine-capped RNA precipitated. Furthermore, RNA B sedimented with pre-rRNAs in nondenaturing sucrose gradients, similarly to U3 and other nucleolar snRNAs, suggesting that RNA B is hydrogen bonded to rRNA intermediates and might be involved in their processing.

Experimental evidence for the secondary structure of the hepatitis delta virus ribozyme.

Specific features of a model for the secondary structure of the self-cleaving RNA sequences (ribozymes) of hepatitis delta virus were rigorously tested. Using a self-cleaving form of the antigenomic sequence, mutations were made in the 5' and 3' sequences of each of four duplex regions within the proposed ribozyme structure. Precursor RNA from each variant sequence was prepared and the kinetics of cleavage in 10 mM Mg2+ at 37 degrees was examined. The data was quantified to determine an end point and a first-order rate constant for cleavage with each mutant by fitting the data to the exponential form of the first-order rate equation. With regard to the final extent of cleavage, most mutations in these regions appeared to have little effect, however, the kinetics indicated that disruption of the potential for basepairing resulted in dramatic decreases in the rate constant for cleavage. These results are consistent with the idea that most of the mutations affected ribozyme activity rather than an equilibrium between precursor and cleavage products. Mutations that reduced rates were compensated by changes that restored the potential for Watson-Crick pairing. Ribonuclease probing of ribozyme variants containing mismatches and compensatory changes allowed direct correlation of structural changes with the mutations. This provided an independent validation of the functional kinetic assay. Thus, site-directed mutagenesis was consistent with a proposed ribozyme secondary structure containing 4 distinct base-paired regions.