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.

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.

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.

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.

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.

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.

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.

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.

Locating chemical modifications in RNA sequences through ribonucleases and LC-MS based analysis.

Knowledge of the structural information is essential for understanding the functional details of modified RNA. Cellular non-coding RNA such as rRNA, tRNA and even viral RNAs contain a number of post-transcriptional modifications with varied degree of diversity and density. In this chapter, we discuss the use of a combination of biochemical and analytical tools such as ribonucleases and liquid chromatography coupled with mass spectrometry approaches for characterization of modified RNA. We present the protocols and alternate strategies for obtaining confident modified sequence information to facilitate the understanding of function.

Oxidative Damage to RNA is Altered by the Presence of Interacting Proteins or Modified Nucleosides.

Oxidative stress triggered by the Fenton reaction (chemical) or UVR exposure (photo) can damage cellular biomolecules including RNA through oxidation of nucleotides. Besides such xenobiotic chemical modifications, RNA also contains several post-transcriptional nucleoside modifications that are installed by enzymes to modulate structure, RNA-protein interactions, and biochemical functions. We examined the extent of oxidative damage to naturally modified RNA which is required for cellular protein synthesis under two different contexts. The extent of oxidative damage is higher when RNA is not associated with proteins, but the degree of damage is lower when the RNA is presented in the form of a ribonucleoprotein complex, such as an intact ribosome. Our studies also indicate that absence of methylations in ribosomal RNA at specific positions could make it more susceptible to photooxidative stress. However, the extent of guanosine oxidation varied with the position at which the modification is deficient, indicating position-dependent structural effects. Further, an E. coli strain deficient in 5-methylaminomethyl-2-thiouridine (mnm5s2U) (found in lysine and glutamate tRNA anticodon) is more vulnerable to oxidative RNA damage compared to its wildtype strain suggesting an auxiliary function for the mnm5s2U modification. These studies indicate that oxidative damage to RNA is altered by the presence of enzymatic modified nucleosides or protein association inside the cell.

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.

Characterization of UVA-Induced Alterations to Transfer RNA Sequences.

Ultraviolet radiation (UVR) adversely affects the integrity of DNA, RNA, and their nucleoside modifications. By employing liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based RNA modification mapping approaches, we identified the transfer RNA (tRNA) regions most vulnerable to photooxidation. Photooxidative damage to the anticodon and variable loop regions was consistently observed in both modified and unmodified sequences of tRNA upon UVA (λ 370 nm) exposure. The extent of oxidative damage measured in terms of oxidized guanosine, however, was higher in unmodified RNA compared to its modified version, suggesting an auxiliary role for nucleoside modifications. The type of oxidation product formed in the anticodon stem-loop region varied with the modification type, status, and whether the tRNA was inside or outside the cell during exposure. Oligonucleotide-based characterization of tRNA following UVA exposure also revealed the presence of novel photoproducts and stable intermediates not observed by nucleoside analysis alone. This approach provides sequence-specific information revealing potential hotspots for UVA-induced damage in tRNAs.

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.

Oligonucleotide analysis by hydrophilic interaction liquid chromatography-mass spectrometry in the absence of ion-pair reagents.

Improving our understanding of nucleic acids, both in biological and synthetic applications, remains a bustling area of research for both academic and industrial laboratories. As nucleic acids research evolves, so must the analytical techniques used to characterize nucleic acids. One powerful analytical technique has been coupled liquid chromatography - tandem mass spectrometry (LC-MS/MS). To date, the most successful chromatographic mode has been ion-pairing reversed-phase liquid chromatography. Hydrophilic interaction liquid chromatography (HILIC), in the absence of ion-pair reagents, has been investigated here as an alternative chromatographic approach to the analysis of oligonucleotides. By combining a mobile phase system using commonly employed in liquid chromatography-mass spectrometry (LC-MS) - i.e., water, acetonitrile, and ammonium acetate - and a new, commercially available diol-based HILIC column, high chromatographic and mass spectrometric performance for a wide range of oligonucleotides is demonstrated. Particular applications of HILIC-MS for the analysis of deoxynucleic acid (DNA) oligomers, modified and unmodified oligoribonucleotides, and phosphorothioate DNA oligonucleotides are presented. Based on the LC-MS performance, this HILIC-based approach provides an attractive, sensitive and robust alternative to prior ion-pairing dependent methods with potential utility for both qualitative and quantitative analyses of oligonucleotides without compromising chromatographic or mass spectrometric performance.

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.

Detection of ribonucleoside modifications by liquid chromatography coupled with mass spectrometry.

A small set of ribonucleoside modifications have been found in different regions of mRNA including the open reading frame. Accurate detection of these specific modifications is critical to understanding their modulatory roles in facilitating mRNA maturation, translation and degradation. While transcriptome-wide next-generation sequencing (NGS) techniques could provide exhaustive information about the sites of one specific or class of modifications at a time, recent investigations strongly indicate cautionary interpretation due to the appearance of false positives. Therefore, it is suggested that NGS-based modification data can only be treated as predicted sites and their existence need to be validated by orthogonal methods. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an analytical technique that can yield accurate and reproducible information about the qualitative and quantitative characteristics of ribonucleoside modifications. Here, we review the recent advancements in LC-MS/MS technology that could help in securing accurate, gold-standard quality information about the resident post-transcriptional modifications of mRNA.

Improving RNA modification mapping sequence coverage by LC-MS through a nonspecific RNase U2-E49A mutant.

We report the identification and use of a mutant of the purine selective ribonuclease RNase U2 that randomly cleaves RNA in a manner that is directly compatible with RNA modification mapping by mass spectrometry. A number of RNase U2 mutants were generated using site-saturation mutagenesis. The enzyme activity and specificity were tested using oligonucleotide substrates, which revealed an RNase U2 E49A mutant with limited specificity and a tendency to undercut RNA. Using this mutant, RNA digestion conditions were optimized to yield long, overlapping digestion products, which improve sequence coverage in RNA modification mapping experiments. The analytical utility of this mutant was demonstrated by liquid chromatography tandem mass spectrometry (LC-MS/MS) mapping of several modified RNAs where 100% sequence coverage could be obtained using only a single enzymatic digestion. This new mutant facilitates more accurate and efficient RNA modification mapping than traditional highly base-specific RNases that are currently used.

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.

Differentiating Positional Isomers of Nucleoside Modifications by Higher-Energy Collisional Dissociation Mass Spectrometry (HCD MS).

The analytical identification of positional isomers (e.g., 3-, N4-, 5-methylcytidine) within the > 160 different post-transcriptional modifications found in RNA can be challenging. Conventional liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) approaches rely on chromatographic separation for accurate identification because the collision-induced dissociation (CID) mass spectra of these isomers nearly exclusively yield identical nucleobase ions (BH2+) from the same molecular ion (MH+). Here, we have explored higher-energy collisional dissociation (HCD) as an alternative fragmentation technique to generate more informative product ions that can be used to differentiate positional isomers. LC-MS/MS of modified nucleosides characterized using HCD led to the creation of structure- and HCD energy-specific fragmentation patterns that generated unique fingerprints, which can be used to identify individual positional isomers even when they cannot be separated chromatographically. While particularly useful for identifying positional isomers, the fingerprinting capabilities enabled by HCD also offer the potential to generate HPLC-independent spectral libraries for the rapid analysis of modified ribonucleosides. Graphical Abstract ᅟ.