Patrick A. Limbach

Transcriptome-wide distribution and function of RNA hydroxymethylcytosine

Chemical modification of RNA for function Chemical modifications play an important role in modifying and regulating the function of DNA and RNA. Delatte et al. show that, in the fruit fly, many messenger RNAs (mRNAs) contain the modified base 5-hydroxymethylcytosine (5hmC). The chemical mark is added by the same enzyme that adds 5hmC to DNA. Because many mRNAs involved in neuronal development contain 5hmC, blocking the enzyme causes brain defects and is lethal. In vivo, RNA hydroxymethylation promotes mRNA translation. Science, this issue p. 282 Posttranscriptional modification of messenger RNAs (mRNAs) is prevalent in Drosophila and promotes mRNA translation. Hydroxymethylcytosine, well described in DNA, occurs also in RNA. Here, we show that hydroxymethylcytosine preferentially marks polyadenylated RNAs and is deposited by Tet in Drosophila. We map the transcriptome-wide hydroxymethylation landscape, revealing hydroxymethylcytosine in the transcripts of many genes, notably in coding sequences, and identify consensus sites for hydroxymethylation. We found that RNA hydroxymethylation can favor mRNA translation. Tet and hydroxymethylated RNA are found to be most abundant in the Drosophila brain, and Tet-deficient fruitflies suffer impaired brain development, accompanied by decreased RNA hydroxymethylation. This study highlights the distribution, localization, and function of cytosine hydroxymethylation and identifies central roles for this modification in Drosophila.

Molecular mass measurement of intact ribonucleic acids via electrospray ionization quadrupole mass spectrometry

The use of electrospray ionization mass spectrometry for the accurate determination of molecular masses of polynucleotides and small nucleic acids is developed. The common problem of gas phase cation adduction that is particularly prevalent in the mass spectrometric analysis of nucleic acids is reduced through the use of ammonium acetate precipitations and by the addition of chemical additives that compete for adduct ions in solution. The addition of chelating agents such as trans-1,2-diaminocyclohexane-N,N,N,′,N′-tetraacetic acid to remove divalent metal ions and triethylamine to displace monovalent cations from the analyte, in conjunction with ammonium acetate precipitation, reduces cation adduction to levels that permit accurate mass analysis (mass errors of less than 0.01%) without further complex cleanup procedures. The potential utility of accurate mass measurements of small ribonucleic acids is discussed.

Agmatidine, a modified cytidine in the anticodon of archaeal tRNA(Ile), base pairs with adenosine but not with guanosine.

Modification of the cytidine in the first anticodon position of the AUA decoding tRNAIle () of bacteria and archaea is essential for this tRNA to read the isoleucine codon AUA and to differentiate between AUA and the methionine codon AUG. To identify the modified cytidine in archaea, we have purified this tRNA species from Haloarcula marismortui, established its codon reading properties, used liquid chromatography–mass spectrometry (LC-MS) to map RNase A and T1 digestion products onto the tRNA, and used LC-MS/MS to sequence the oligonucleotides in RNase A digests. These analyses revealed that the modification of cytidine in the anticodon of adds 112 mass units to its molecular mass and makes the glycosidic bond unusually labile during mass spectral analyses. Accurate mass LC-MS and LC-MS/MS analysis of total nucleoside digests of the demonstrated the absence in the modified cytidine of the C2-oxo group and its replacement by agmatine (decarboxy-arginine) through a secondary amine linkage. We propose the name agmatidine, abbreviation C+, for this modified cytidine. Agmatidine is also present in Methanococcus maripaludis and in Sulfolobus solfataricus total tRNA, indicating its probable occurrence in the AUA decoding tRNAIle of euryarchaea and crenarchaea. The identification of agmatidine shows that bacteria and archaea have developed very similar strategies for reading the isoleucine codon AUA while discriminating against the methionine codon AUG.

Mononucleotide gas-phase proton affinities as determined by the kinetic method

The goal of this work is to determine the proton affinities of (deoxy)nucleoside 5′- and 3′-monophosphates (mononucleotides) using the kinetic method with fast atom bombardment mass spectrometry. The proton affinities of the (deoxy)nucleoside 5′- and 3′-monophosphates yielded the following trend: (deoxy)adenosine monophosphates > (deoxy)guanosine monophosphates > (deoxy)cytidine monophosphates ≫ deoxythymidine/uridine monophosphates. In all cases the proton affinity decreases or remains the same with the addition of the phosphate group from those values reported for nucleosides. The proton affinity is dependent on the location of the phosphate backbone (5′- vs. 3′-phosphates): the 3′-monophosphates have lower proton affinities than the 5′-monophosphates except for the thymidine/uridine monophosphates where the trend is reversed. Molecular modeling was utilized to determine if multiple protonation sites and intramolecular hydrogen bond formation would influence the proton affinity measurements. Semiempirical calculations of the proton affinities at various locations on each mononucleotide were performed and compared to the experimental results. The possible influence of intramolecular hydrogen bonding between the nucleobases and the phosphate group on the measured and calculated proton affinities is discussed.

Characterization of oligonucleotides and nucleic acids by mass spectrometry.

The continued refinement of two recent methods for producing gas-phase ions, electrospray ionization and matrix-assisted laser desorption ionization, has resulted in new techniques for the rapid characterization of oligonucleotides by mass spectrometry. Using commercially available instruments, molecular mass measurements at the 20-mer level, with errors less than 2 Da, can now be made routinely in less than 15 min. Progress has also been achieved in the development of mass spectrometry for rapid sequencing of oligonucleotides smaller than 25 residues.

Bisphenol A facilitates bypass of androgen ablation therapy in prostate cancer

Prostatic adenocarcinomas depend on androgen for growth and survival. First line treatment of disseminated disease exploits this dependence by specifically targeting androgen receptor function. Clinical evidence has shown that androgen receptor is reactivated in recurrent tumors despite the continuance of androgen deprivation therapy. Several factors have been shown to restore androgen receptor activity under these conditions, including somatic mutation of the androgen receptor ligand-binding domain. We have shown previously that select tumor-derived mutants of the androgen receptor are receptive to activation by bisphenol A (BPA), an endocrine-disrupting compound that is leached from polycarbonate plastics and epoxy resins into the human food supply. Moreover, we have shown that BPA can promote cell cycle progression in cultured prostate cancer cells under conditions of androgen deprivation. Here, we challenged the effect of BPA on the therapeutic response in a xenograft model system of prostate cancer containing the endogenous BPA-responsive AR-T877A mutant protein. We show that after androgen deprivation, BPA enhanced both cellular proliferation rates and tumor growth. These effects were mediated, at least in part, through androgen receptor activity, as prostate-specific antigen levels rose with accelerated kinetics in BPA-exposed animals. Thus, at levels relevant to human exposure, BPA can modulate tumor cell growth and advance biochemical recurrence in tumors expressing the AR-T877A mutation. [Mol Cancer Ther 2006;5(12):3181–90]

Electrospray ionization mass spectrometry of metalloporphyrins

The magnesium, nickel, copper, zinc and vanadium metalloporphyrins from octaethylporphyrin, etioporphyrin I and tetraphenylporphyrin were characterized using electrospray ionization mass spectrometry (ESI-MS). The ion abundance of each of the porphyrins present in binary mixtures was monitored as a function of the porphyrin concentration and is dependent on the metalloporphyrin oxidation potential. It was found that, for binary mixtures of metalloporphyrins whose oxidation potentials differ by less than 0.1 V, the resulting ion abundance of each species is directly proportional to the concentration of each analyte in the mixture. For binary mixtures whose oxidation potentials differ by more than 0.1 V, relative abundances of the radical cations of each metalloporphyrin are determined by the oxidation potential and concentration of each metalloporphyrin with the analyte of lowest oxidation potential being ionized preferentially. The ability to ionize selectively one porphyrin over another in a binary mixture offers the potential to use ESI-MS for the qualitative analysis of porphyrins present in complex mixtures.

Influence of ionization energy on charge-transfer ionization in matrix-assisted laser desorption/ionization mass spectrometry

Abstract In this study, non-polar matrices are used in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to analyze selected non-polar analytes. Our hypothesis is that gas-phase charge-transfer reactions between matrix and analyte are responsible for the generation of analyte radical molecular ions. Following this hypothesis, the ionization energies of the matrices and analytes should have a direct influence on the production of radical molecular cations of the analytes. Several non-polar analytes, including ferrocene and ferrocene derivatives, trans-stilbene, triphenylphosphine, 2,2′-methylenebis(6-tert-butyl-4-methylphenol), biphenyl and 1,4-bis(methylthio)benzene were studied using positive-ion mode MALDI-TOFMS. The results of these studies demonstrate that formation of the radical molecular cation depends on the difference in ionization energies between the matrix and the analyte. The propensity for charge-transfer ionization, as opposed to proton-transfer ionization, for these analytes, was confirmed using atmospheric pressure chemical ionization mass spectrometry. Charge-transfer ionization using non-polar matrices in MALDI-MS is a suitable method for the characterization of a number of non-polar, thermally labile analytes.

Structural and Mechanistic Basis for Enhanced Translational Efficiency by 2-Thiouridine at the tRNA Anticodon Wobble Position

The 2-thiouridine (s(2)U) at the wobble position of certain bacterial and eukaryotic tRNAs enhances aminoacylation kinetics, assists proper codon-anticodon base pairing at the ribosome A-site, and prevents frameshifting during translation. By mass spectrometry of affinity-purified native Escherichia coli tRNA1(Gln)UUG, we show that the complete modification at the wobble position 34 is 5-carboxyaminomethyl-2-thiouridine (cmnm(5)s(2)U). The crystal structure of E. coli glutaminyl-tRNA synthetase (GlnRS) bound to native tRNA1(Gln) and ATP demonstrates that cmnm(5)s(2)U34 improves the order of a previously unobserved 11-amino-acid surface loop in the distal β-barrel domain of the enzyme and imparts other local rearrangements of nearby amino acids that create a binding pocket for the 2-thio moiety. Together with previously solved structures, these observations explain the degenerate recognition of C34 and modified U34 by GlnRS. Comparative pre-steady-state aminoacylation kinetics of native tRNA1(Gln), synthetic tRNA1(Gln) containing s(2)U34 as sole modification, and unmodified wild-type and mutant tRNA1(Gln) and tRNA2(Gln) transcripts demonstrates that the exocyclic sulfur moiety improves tRNA binding affinity to GlnRS 10-fold compared with the unmodified transcript and that an additional fourfold improvement arises from the presence of the cmnm(5) moiety. Measurements of Gln-tRNA(Gln) interactions at the ribosome A-site show that the s(2)U modification enhances binding affinity to the glutamine codons CAA and CAG and increases the rate of GTP hydrolysis by E. coli EF-Tu by fivefold.

Gas-phase hydrogen/deuterium exchange of positively charged mononucleotides by use of Fourier-transform ion cyclotron resonance mass spectrometry.

The gas-phase structures of protonated (deoxy)nucleoside-5′- and 3′-monophosphates (mononucleotides) have been examined by the use of gas-phase hydrogen/deuterium (H/D) exchange and high-field Fourier-transform ion cyclotron resonance mass spectrometry. These nucleotides were reacted with three different deuterating reagents: ND3, D2O, and D2S, of which ND3 was the most effective. All mononucleotides fully exchanged their labile hydrogen for deuterium with ND3 with the exception of deoxycytidine-3′-monophosphate, deoxyadenosine-5′-monophosphate, adenosine-5′-monophosphate, and adenosine-3′-monophosphate. Semiempirical calculations demonstrate the presence of hydrogen bonding upon protonation of the purine mononucleotides which may lead to incomplete H/D exchange. H/D exchange rates differed between the deoxymononucleotides and the ribomononucleotides, suggesting that the 2′-OH group plays an important role in the exchange process. Reactions of nucleosides and mononucleotides with D2O demonstrate that a structure-specific long-lived ion—molecule complex between D2O and the mononucleotide involving the phosphate group is necessary for exchange to overcome the high-energy activation barrier. In contrast, a structure-specific long-lived ion—molecule complex between the mononucleotides and ND3 is not required for exchange to occur.