Identification and mapping of post-transcriptional modifications on the HIV-1 antisense transcript Ast in human cells.

The human immunodeficiency virus type 1 (HIV-1) encodes multiple RNA molecules. Transcripts that originate from the proviral 5' long terminal repeat (LTR) function as messenger RNAs for the expression of 16 different mature viral proteins. In addition, HIV-1 expresses an antisense transcript (Ast) from the 3'LTR, which has both protein-coding and noncoding properties. While the mechanisms that regulate the coding and noncoding activities of Ast remain unknown, post-transcriptional modifications are known to influence RNA stability, interaction with protein partners, and translation capacity. Here, we report the nucleoside modification profile of Ast obtained through liquid chromatography coupled with mass spectrometry (LC-MS) analysis. The epitranscriptome includes a limited set of modified nucleosides but predominantly ribose methylations. A number of these modifications were mapped to specific positions of the sequence through RNA modification mapping procedures. The presence of modifications on Ast is consistent with the RNA-modifying enzymes interacting with Ast The identification and mapping of Ast post-transcriptional modifications is expected to elucidate the mechanisms through which this versatile molecule can carry out diverse activities in different cell compartments. Manipulation of post-transcriptional modifications on the Ast RNA may have therapeutic implications.

Higher-Energy Collisional Dissociation Mass Spectral Networks for the Rapid, Semi-automated Characterization of Known and Unknown Ribonucleoside Modifications.

Higher-energy collisional dissociation (HCD) of modified ribonucleosides generates characteristic and highly reproducible nucleoside-specific tandem mass spectra (MS/MS). Here, we demonstrate the capability of HCD spectra in combination with spectral matching for the semi-automated characterization of ribonucleosides. This process involved the generation of an HCD spectral library and the establishment of a mass spectral network for rapid detection with high sensitivity and specificity in a retention time-independent fashion. Systematic spectral matching analysis of the MS/MS spectra of tRNA hydrolysates from different organisms has helped us to uncover evidence for the existence of novel ribonucleoside modifications such as s2Cm and OHyW-14. Such an untargeted label-free approach has the potential to be integrated with other methods, including those that use isotope labeling, to simplify the characterization of unknown modified ribonucleosides. These findings suggest the compilation of a universal spectral network, for the characterization of known and unknown ribonucleosides, could accelerate discoveries in the epitranscriptome.

RNA Cleavage Properties of Nucleobase-Specific RNase MC1 and Cusativin Are Determined by the Dinucleotide-Binding Interactions in the Enzyme-Active Site.

Knowledge of the cleavage specificity of ribonucleases is critical for their application in RNA modification mapping or RNA-protein binding studies. Here, we detail the cleavage specificity and efficiency of ribonuclease MC1 and cusativin using a customized RNA sequence that contained all dinucleotide combinations and homopolymer sequences. The sequencing of the oligonucleotide digestion products by a semi-quantitative liquid chromatography coupled with mass spectrometry (LC-MS) analysis documented as little as 0.5-1% cleavage levels for a given dinucleotide sequence combination. While RNase MC1 efficiently cleaved the [A/U/C]pU dinucleotide bond, no cleavage was observed for the GpU bond. Similarly, cusativin efficiently cleaved Cp[U/A/G] dinucleotide combinations along with UpA and [A/U]pU, suggesting a broader specificity of dinucleotide preferences. The molecular interactions between the substrate and active site as determined by the dinucleotide docking studies of protein models offered additional evidence and support for the observed substrate specificity. Targeted alteration of the key amino acid residues in the nucleotide-binding site confirms the utility of this in silico approach for the identification of key interactions. Taken together, the use of bioanalytical and computational approaches, involving LC-MS and ligand docking of tertiary structural models, can form a powerful combination to help explain the RNA cleavage behavior of RNases.

Chemical Amination/Imination of Carbonothiolated Nucleosides During RNA Hydrolysis.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the gold-standard technique to study RNA and its various modifications. While most research on RNA nucleosides has been focused on their biological roles, discovery of new modifications remains of interest. With state-of-the-art technology, the presence of artifacts can confound the identification of new modifications. Here, we report the characterization of a non-natural mcm5 isoC ribonucleoside in S. cerevisiae total tRNA hydrolysate by higher-energy collisional dissociation (HCD)-based fingerprints and isotope labeling of RNA. Its discovery revealed a class of amino/imino ribonucleoside artifacts that are generated during RNA hydrolysis under ammonium-buffered mild basic conditions. We then identified digestion conditions that can reduce or eliminate their formation. These finding and method enhancements will improve the accurate detection of new RNA modifications.

Improved RNA modification mapping of cellular non-coding RNAs using C- and U-specific RNases.

Locating ribonucleoside modifications within an RNA sequence requires digestion of the RNA into oligoribonucleotides of amenable size for subsequent analysis by LC-MS (liquid chromatography-mass spectrometry). This approach, widely referred to as RNA modification mapping, is facilitated through ribonucleases (RNases) such as T1 (guanosine-specific), U2 (purine-selective) and A (pyrimidine-specific) among others. Sequence coverage by these enzymes depends on positioning of the recognized nucleobase (such as guanine or purine or pyrimidine) in the sequence and its ribonucleotide composition. Using E. coli transfer RNA (tRNA) and ribosomal RNA (rRNA) as model samples, we demonstrate the ability of complementary nucleobase-specific ribonucleases cusativin (C-specific) and MC1 (U-specific) to generate digestion products that facilitate confident mapping of modifications in regions such as G-rich and pyrimidine-rich segments of RNA, and to distinguish C to U sequence differences. These enzymes also increase the number of oligonucleotide digestion products that are unique to a specific RNA sequence. Further, with these additional RNases, multiple modifications can be localized with high confidence in a single set of experiments with minimal dependence on the individual tRNA abundance in a mixture. The sequence overlaps observed with these complementary digestion products and that of RNase T1 improved sequence coverage to 75% or above. A similar level of sequence coverage was also observed for the 2904 nt long 23S rRNA indicating their utility has no dependence on RNA size. Wide-scale adoption of these additional modification mapping tools could help expedite the characterization of modified RNA sequences to understand their structural and functional role in various living systems.

A metastable rRNA junction essential for bacterial 30S biogenesis.

Tertiary sequence motifs encode interactions between RNA helices that create the three-dimensional structures of ribosomal subunits. A Right Angle motif at the junction between 16S helices 5 and 6 (J5/6) is universally conserved amongst small subunit rRNAs and forms a stable right angle in minimal RNAs. J5/6 does not form a right angle in the mature ribosome, suggesting that this motif encodes a metastable structure needed for ribosome biogenesis. In this study, J5/6 mutations block 30S ribosome assembly and 16S maturation in Escherichia coli. Folding assays and in-cell X-ray footprinting showed that J5/6 mutations favor an assembly intermediate of the 16S 5' domain and prevent formation of the central pseudoknot. Quantitative mass spectrometry revealed that mutant pre-30S ribosomes lack protein uS12 and are depleted in proteins uS5 and uS2. Together, these results show that impaired folding of the J5/6 right angle prevents the establishment of inter-domain interactions, resulting in global collapse of the 30S structure observed in electron micrographs of mutant pre-30S ribosomes. We propose that the J5/6 motif is part of a spine of RNA helices that switch conformation at distinct stages of assembly, linking peripheral domains with the 30S active site to ensure the integrity of 30S biogenesis.

tRNA Modification Profiles and Codon-Decoding Strategies in Methanocaldococcus jannaschii.

tRNAs play a critical role in mRNA decoding, and posttranscriptional modifications within tRNAs drive decoding efficiency and accuracy. The types and positions of tRNA modifications in model bacteria have been extensively studied, and tRNA modifications in a few eukaryotic organisms have also been characterized and localized to particular tRNA sequences. However, far less is known regarding tRNA modifications in archaea. While the identities of modifications have been determined for multiple archaeal organisms, Haloferax volcanii is the only organism for which modifications have been extensively localized to specific tRNA sequences. To improve our understanding of archaeal tRNA modification patterns and codon-decoding strategies, we have used liquid chromatography and tandem mass spectrometry to characterize and then map posttranscriptional modifications on 34 of the 35 unique tRNA sequences of Methanocaldococcus jannaschii A new posttranscriptionally modified nucleoside, 5-cyanomethyl-2-thiouridine (cnm5s2U), was discovered and localized to position 34. Moreover, data consistent with wyosine pathway modifications were obtained beyond the canonical tRNAPhe as is typical for eukaryotes. The high-quality mapping of tRNA anticodon loops enriches our understanding of archaeal tRNA modification profiles and decoding strategies.IMPORTANCE While many posttranscriptional modifications in M. jannaschii tRNAs are also found in bacteria and eukaryotes, several that are unique to archaea were identified. By RNA modification mapping, the modification profiles of M. jannaschii tRNA anticodon loops were characterized, allowing a comparative analysis with H. volcanii modification profiles as well as a general comparison with bacterial and eukaryotic decoding strategies. This general comparison reveals that M. jannaschii, like H. volcanii, follows codon-decoding strategies similar to those used by bacteria, although position 37 appears to be modified to a greater extent than seen in H. volcanii.

Pseudouridine in the Anticodon of Escherichia coli tRNATyr(QΨA) Is Catalyzed by the Dual Specificity Enzyme RluF.

Pseudouridine is found in almost all cellular ribonucleic acids (RNAs). Of the multiple characteristics attributed to pseudouridine, making messenger RNAs (mRNAs) highly translatable and non-immunogenic is one such feature that directly implicates this modification in protein synthesis. We report the existence of pseudouridine in the anticodon of Escherichia coli tyrosine transfer RNAs (tRNAs) at position 35. Pseudouridine was verified by multiple detection methods, which include pseudouridine-specific chemical derivatization and gas phase dissociation of RNA during liquid chromatography tandem mass spectrometry (LC-MS/MS). Analysis of total tRNA isolated from E. coli pseudouridine synthase knock-out mutants identified RluF as the enzyme responsible for this modification. Furthermore, the absence of this modification compromises the translational ability of a luciferase reporter gene coding sequence when it is preceded by multiple tyrosine codons. This effect has implications for the translation of mRNAs that are rich in tyrosine codons in bacterial expression systems.

Novel ribonuclease activity of cusativin from Cucumis sativus for mapping nucleoside modifications in RNA.

A recombinant ribonuclease, cusativin, was characterized for its cytidine-specific cleavage ability of RNA to map chemical modifications. Following purification of native cusativin protein as described before (Rojo et al. Planta 194:328, 17), partial amino acid sequencing was carried out to identify the corresponding protein coding gene in cucumber genome. Cloning and heterologous expression of the identified gene in Escherichia coli resulted in successful production of active protein as a C-terminal His-tag fusion protein. The ribonuclease activity and cleavage specificity of the fusion protein were confirmed with a variety of tRNA isoacceptors and total tRNA. Characterization of cusativin digestion products by ion-pairing reverse-phase liquid chromatography coupled with mass spectrometry (IP-RP-LC-MS) analysis revealed cleavage of CpA, CpG, and CpU phosphodiester bonds at the 3'-terminus of cytidine under optimal digestion conditions. Ribose methylation or acetylation of cytosine inhibited RNA cleavage. The CpC phosphodiester bond was also resistant to cusativin-mediated RNA cleavage; a feature to our knowledge has not been reported for other nucleobase-specific ribonucleases. Here, we demonstrate the analytical utility of such a novel feature for obtaining high-sequence coverage and accurate mapping of modified residues in substrate RNAs. Graphical abstract Cytidine-specific novel ribonuclease activity of cusativin.

An Arabidopsis ATP-dependent, DEAD-box RNA helicase loses activity upon IsoAsp formation but is restored by PROTEIN ISOASPARTYL METHYLTRANSFERASE.

Orthodox seeds are capable of withstanding severe dehydration. However, in the dehydrated state, Asn and Asp residues in proteins can convert to succinimide residues that can further react to predominantly form isomerized isoAsp residues upon rehydration (imbibition). IsoAsp residues can impair protein function and can render seeds nonviable, but PROTEIN ISOASPARTYL METHYLTRANSFERASE (PIMT) can initiate isoAsp conversion to Asp residues. The proteins necessary for translation upon imbibition in orthodox seeds may be particularly important to maintain in an active state. One such protein is the large, multidomain protein, Arabidopsis thaliana PLANT RNA HELICASE75 (PRH75), a DEAD-box helicase known to be susceptible to isoAsp residue accumulation. However, the consequences of such isomerization on PRH75 catalysis and for the plant are unknown. Here, it is demonstrated that PRH75 is necessary for successful seed development. It acquires isoAsp rapidly during heat stress, which eliminates RNA unwinding (but not rewinding) competence. The repair by PIMT is able to restore PRH75's complex biochemical activity provided isoAsp formation has not led to subsequent, destabilizing conformational alterations. For PRH75, an important enzymatic activity associated with translation would be eliminated unless rapidly repaired by PIMT prior to additional, deleterious conformational changes that would compromise seed vitality and germination.

Bioengineering Novel Chimeric microRNA-34a for Prodrug Cancer Therapy: High-Yield Expression and Purification, and Structural and Functional Characterization.

Development of anticancer treatments based on microRNA (miRNA/miR) such as miR-34a replacement therapy is limited to the use of synthetic RNAs with artificial modifications. Herein, we present a new approach to a high-yield and large-scale biosynthesis, in Escherichia coli using transfer RNA (tRNA) scaffold, of chimeric miR-34a agent, which may act as a prodrug for anticancer therapy. The recombinant tRNA fusion pre-miR-34a (tRNA/mir-34a) was quickly purified to a high degree of homogeneity (>98%) using anion-exchange fast protein liquid chromatography, whose primary sequence and post-transcriptional modifications were directly characterized by mass spectrometric analyses. Chimeric tRNA/mir-34a showed a favorable cellular stability while it was degradable by several ribonucleases. Deep sequencing and quantitative real-time polymerase chain reaction studies revealed that tRNA-carried pre-miR-34a was precisely processed to mature miR-34a within human carcinoma cells, and the same tRNA fragments were produced from tRNA/mir-34a and the control tRNA scaffold (tRNA/MSA). Consequently, tRNA/mir-34a inhibited the proliferation of various types of human carcinoma cells in a dose-dependent manner and to a much greater degree than the control tRNA/MSA, which was mechanistically attributable to the reduction of miR-34a target genes. Furthermore, tRNA/mir-34a significantly suppressed the growth of human non-small-cell lung cancer A549 and hepatocarcinoma HepG2 xenograft tumors in mice, compared with the same dose of tRNA/MSA. In addition, recombinant tRNA/mir-34a had no or minimal effect on blood chemistry and interleukin-6 level in mouse models, suggesting that recombinant RNAs were well tolerated. These findings provoke a conversation on producing biologic miRNAs to perform miRNA actions, and point toward a new direction in developing miRNA-based therapies.

Chimeric MicroRNA-1291 Biosynthesized Efficiently in Escherichia coli Is Effective to Reduce Target Gene Expression in Human Carcinoma Cells and Improve Chemosensitivity.

In contrast to the growing interests in studying noncoding RNAs (ncRNAs) such as microRNA (miRNA or miR) pharmacoepigenetics, there is a lack of efficient means to cost effectively produce large quantities of natural miRNA agents. Our recent efforts led to a successful production of chimeric pre-miR-27b in bacteria using a transfer RNA (tRNA)-based recombinant RNA technology, but at very low expression levels. Herein, we present a high-yield expression of chimeric pre-miR-1291 in common Escherichia coli strains using the same tRNA scaffold. The tRNA fusion pre-miR-1291 (tRNA/mir-1291) was then purified to high homogeneity using affinity chromatography, whose primary sequence and post-transcriptional modifications were directly characterized by mass spectrometric analyses. Chimeric tRNA/mir-1291 was readily processed to mature miR-1291 in human carcinoma MCF-7 and PANC-1 cells. Consequently, recombinant tRNA/mir-1291 reduced the protein levels of miR-1291 target genes, including ABCC1, FOXA2, and MeCP2, as compared with cells transfected with the same doses of control methionyl-tRNA scaffold with a sephadex aptamer (tRNA/MSA). In addition, tRNA-carried pre-miR-1291 suppressed the growth of MCF-7 and PANC-1 cells in a dose-dependent manner, and significantly enhanced the sensitivity of ABCC1-overexpressing PANC-1 cells to doxorubicin. These results indicate that recombinant miR-1291 agent is effective in the modulation of target gene expression and chemosensitivity, which may provide insights into high-yield bioengineering of new ncRNA agents for pharmacoepigenetics research.

Fast and efficient size exclusion chromatography of adeno associated viral vectors with 2.5 micrometer particle low adsorption columns.

More and more transformative gene therapies (GTx) are reaching commercialization stage and many of them use Adeno Associated Viruses (AAVs) as their vector. Being larger than therapeutic antibodies, their size variant analysis poses an analytical challenge that must be addressed to speed up the development processes. Size exclusion chromatography (SEC) can provide critical information on the quality and purity of the product, but its full potential is not yet utilized by currently applied columns that are (i) packed with relatively large particles, (ii) prepared exclusively in large formats and (iii) built using metal hardware that is prone to secondary interactions. In this paper, we investigate the use of state-of-the-art sub-3 µm particles to address existing limitations. A prototype 2.5 µm column was found to deliver superior kinetic efficiency, significant reduction in run times and increased resolution of separations. No evidence for shear or sample sieving effects were found during comparisons with conventional 5 µm columns. Moreover, use of low adsorption hardware enabled the application of a wide range of mobile phase conditions and a chance to apply a more robust platform method for several AAV serotypes. The resulting method was tested for its reproducibility as well as utility for critical quality attribute assays, including multiangle light scattering based (MALS) measurements of size and molar mass. Thus, a new tool for higher resolution, higher throughput size variant analysis of AAVs has been described.

Mapping m6A Sites on HIV-1 RNA Using Oligonucleotide LC-MS/MS.

The biological significance of chemical modifications to the ribonucleic acid (RNA) of human immunodeficiency virus type-1 (HIV-1) has been recognized. However, our understanding of the site-specific and context-dependent roles of these chemical modifications remains limited, primarily due to the absence of nucleotide-resolution mapping of modification sites. In this study, we present a method for achieving nucleotide-resolution mapping of chemical modification sites on HIV-1 RNA using liquid chromatography and tandem mass spectrometry (LC-MS/MS). LC-MS/MS, a powerful tool capable of directly analyzing native RNAs, has proven effective for mapping RNA modifications in small RNA molecules, including ribosomal RNA and transfer RNA. However, longer RNAs have posed challenges, such as the 9 Kb HIV-1 virion RNA, due to the complexity of and ambiguity in mass differences among RNase T1-cleaved RNA fragments in LC-MS/MS data. Here, we introduce a new target RNA enrichment method to isolate small local RNA fragments of HIV-1 RNA that potentially harbor site-specific N6-methyladenosine (m6A) modifications. In our initial trial, we used target-specific DNA probes only and encountered insufficient RNA fragmentation due to inefficient S1 digestion near the target site. Recognizing that inefficient S1 digestion by HIV-1 RNA is likely due to the formation of secondary structures in proximity to the target site, we designed multiple DNA probes annealing to various sites of HIV-1 RNA to better control the structures of RNA substrates for S1 digestion. The use of these non-target DNA probes significantly improved the isolation of more homogeneous target RNA fragments of approximately 50 bases in length. Oligonucleotide LC-MS/MS analysis of these isolated target RNA fragments successfully separated and detected both m6A-methylated and non-methylated oligomers at the two m6A-predicted sites. The principle of this new target enrichment strategy holds promise and should be broadly applicable to the analysis of any lengthy RNA that was previously deemed infeasible for investigation using oligonucleotide LC-MS/MS.

Single-molecule epitranscriptomic analysis of full-length HIV-1 RNAs reveals functional roles of site-specific m6As.

Although the significance of chemical modifications on RNA is acknowledged, the evolutionary benefits and specific roles in human immunodeficiency virus (HIV-1) replication remain elusive. Most studies have provided only population-averaged values of modifications for fragmented RNAs at low resolution and have relied on indirect analyses of phenotypic effects by perturbing host effectors. Here we analysed chemical modifications on HIV-1 RNAs at the full-length, single RNA level and nucleotide resolution using direct RNA sequencing methods. Our data reveal an unexpectedly simple HIV-1 modification landscape, highlighting three predominant N6-methyladenosine (m6A) modifications near the 3' end. More densely installed in spliced viral messenger RNAs than in genomic RNAs, these m6As play a crucial role in maintaining normal levels of HIV-1 RNA splicing and translation. HIV-1 generates diverse RNA subspecies with distinct m6A ensembles, and maintaining multiple of these m6As on its RNAs provides additional stability and resilience to HIV-1 replication, suggesting an unexplored viral RNA-level evolutionary strategy.

Functional role of methylation of G518 of the 16S rRNA 530 loop by GidB in Mycobacterium tuberculosis.

Posttranscriptional modifications of bacterial rRNA serve a variety of purposes, from stabilizing ribosome structure to preserving its functional integrity. Here, we investigated the functional role of one rRNA modification in particular-the methylation of guanosine at position 518 (G518) of the 16S rRNA in Mycobacterium tuberculosis. Based on previously reported evidence that G518 is located 5 Å; from proline 44 of ribosomal protein S12, which interacts directly with the mRNA wobble position of the codon:anticodon helix at the A site during translation, we speculated that methylation of G518 affects protein translation. We transformed reporter constructs designed to probe the effect of functional lesions at one of the three codon positions on translational fidelity into the wild-type strain, H37Rv, and into a ΔgidB mutant, which lacks the methyltransferase (GidB) that methylates G518. We show that mistranslation occurs less in the ΔgidB mutant only in the construct bearing a lesion in the wobble position compared to H37Rv. Thus, the methylation of G518 allows mistranslation to occur at some level in order for translation to proceed smoothly and efficiently. We also explored the role of methylation at G518 in altering the susceptibility of M. tuberculosis to streptomycin (SM). Using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), we confirmed that G518 is not methylated in the ΔgidB mutant. Furthermore, isothermal titration calorimetry experiments performed on 70S ribosomes purified from wild-type and ΔgidB mutant strains showed that methylation significantly enhances SM binding. These results provide a mechanistic explanation for the low-level, SM-resistant phenotype observed in M. tuberculosis strains that contain a gidB mutation.

Characterizing Benzo[a]pyrene Adducts in Transfer RNAs Using Liquid Chromatography Coupled with Tandem Mass Spectrometry (LC-MS/MS).

The activated forms of the environmental pollutant benzo[a]pyrene (B[a]P), such as benzo[a]pyrene diol epoxide (BPDE), are known to cause damage to genomic DNA and proteins. However, the impact of BPDE on ribonucleic acid (RNA) remains unclear. To understand the full spectrum of potential BPDE-RNA adducts formed, we reacted ribonucleoside standards with BPDE and characterized the reaction products using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). To understand the potential types of adducts that could form with biological RNAs, eukaryotic transfer RNAs (tRNAs) were also reacted with BPDE. The isolation and analysis of the modified and adducted ribonucleosides using LC-MS/MS revealed several BPDE derivatives of post-transcriptional modifications. The approach outlined in this work enables the identification of RNA adducts from BPDE, which can pave the way for understanding the potential impacts of such adducts on the higher-order structure and function of modified RNAs.

Tyrosine transfer RNA levels and modifications during blood-feeding and vitellogenesis in the mosquito, Aedes aegypti.

Mosquitoes such as Aedes aegypti must consume a blood meal for the nutrients necessary for egg production. Several transcriptome and proteome changes occur post blood meal that likely corresponds with codon usage alterations. Transfer RNA (tRNA) is the adapter molecule that reads messenger RNA (mRNA) codons to add the appropriate amino acid during protein synthesis. Chemical modifications to tRNA enhance codons' decoding, improving the accuracy and efficiency of protein synthesis. Here, we examined tRNA modifications and transcripts associated with the blood meal and subsequent periods of vitellogenesis in A. aegypti. More specifically, we assessed tRNA transcript abundance and modification levels in the fat body at critical times post blood-feeding. Based on a combination of alternative codon usage and identification of particular modifications, we identified that increased transcription of tyrosine tRNAs is likely critical during the synthesis of egg yolk proteins in the fat body following a blood meal. Altogether, changes in both the abundance and modification of tRNA are essential factors in the process of vitellogenin production after blood-feeding in mosquitoes.

Ionizing radiation and chemical oxidant exposure impacts on Cryptococcus neoformans transfer RNAs.

Cryptococcus neoformans is a fungus that is able to survive abnormally high levels of ionizing radiation (IR). The radiolysis of water by IR generates reactive oxygen species (ROS) such as H2O2 and OH-. C. neoformans withstands the damage caused by IR and ROS through antioxidant production and enzyme-catalyzed breakdown of ROS. Given these particular cellular protein needs, questions arise whether transfer ribonucleic acids molecules (tRNAs) undergo unique chemical modifications to maintain their structure, stability, and/or function under such environmental conditions. Here, we investigated the effects of IR and H2O2 exposure on tRNAs in C. neoformans. We experimentally identified the modified nucleosides present in C. neoformans tRNAs and quantified changes in those modifications upon exposure to oxidative conditions. To better understand these modified nucleoside results, we also evaluated tRNA pool composition in response to the oxidative conditions. We found that regardless of environmental conditions, tRNA modifications and transcripts were minimally affected. A rationale for the stability of the tRNA pool and its concomitant profile of modified nucleosides is proposed based on the lack of codon bias throughout the C. neoformans genome and in particular for oxidative response transcripts. Our findings suggest that C. neoformans can rapidly adapt to oxidative environments as mRNA translation/protein synthesis are minimally impacted by codon bias.

Removal of 3'-phosphate group by bacterial alkaline phosphatase improves oligonucleotide sequence coverage of RNase digestion products analyzed by collision-induced dissociation mass spectrometry.

RNase mapping by nucleobase-specific endonucleases combined with liquid chromatography/tandem mass spectrometry (LC/MS/MS) is a powerful analytical method for characterizing ribonucleic acids (RNAs). Endonuclease digestion of RNA yields products that contain a 3'-terminal phosphate group. MS/MS via collision-induced dissociation (CID) of these digestion products on a linear ion trap generates fragmentation pathways that include the loss of phosphoric acid (-H(3)PO(4); -98 u), which does not provide information about the sequence of the digestion products and can reduce ion abundance from other pathways that provide sequence information. Here we investigate the use of bacterial alkaline phosphatase (BAP) after RNase digestion to remove the 3'-terminal phosphate from all RNase digestion products prior to LC/MS/MS analysis. RNase digestion products lacking the 3'-phosphate were found to produce CID spectra with more consistent, high-abundance c- and y-type fragment ions as well as significantly more a-Base and w-type ions than digestion products retaining the 3'-phosphate. In this manner, RNase mapping with LC/MS/MS can provide more complete RNA sequence information from fragment ions of higher abundance that are easier to interpret and identify.