Conserved 5-methyluridine tRNA modification modulates ribosome translocation.

While the centrality of posttranscriptional modifications to RNA biology has long been acknowledged, the function of the vast majority of modified sites remains to be discovered. Illustrative of this, there is not yet a discrete biological role assigned for one of the most highly conserved modifications, 5-methyluridine at position 54 in tRNAs (m5U54). Here, we uncover contributions of m5U54 to both tRNA maturation and protein synthesis. Our mass spectrometry analyses demonstrate that cells lacking the enzyme that installs m5U in the T-loop (TrmA in Escherichia coli, Trm2 in Saccharomyces cerevisiae) exhibit altered tRNA modification patterns. Furthermore, m5U54-deficient tRNAs are desensitized to small molecules that prevent translocation in vitro. This finding is consistent with our observations that relative to wild-type cells, trm2Δ cell growth and transcriptome-wide gene expression are less perturbed by translocation inhibitors. Together our data suggest a model in which m5U54 acts as an important modulator of tRNA maturation and translocation of the ribosome during protein synthesis.

Adenosine modifications impede SARS-CoV-2 RNA-dependent RNA transcription.

SARS-CoV-2, the causative virus of the COVID-19 pandemic, follows SARS and MERS as recent zoonotic coronaviruses causing severe respiratory illness and death in humans. The recurrent impact of zoonotic coronaviruses demands a better understanding of their fundamental molecular biochemistry. Nucleoside modifications, which modulate many steps of the RNA life cycle, have been found in SARS-CoV-2 RNA, although whether they confer a pro- or antiviral effect is unknown. Regardless, the viral RNA-dependent RNA polymerase will encounter these modifications as it transcribes through the viral genomic RNA. We investigated the functional consequences of nucleoside modification on the pre-steady state kinetics of SARS-CoV-2 RNA-dependent RNA transcription using an in vitro reconstituted transcription system with modified RNA templates. Our findings show that N 6-methyladenosine and 2'-O-methyladenosine modifications slow the rate of viral transcription at magnitudes specific to each modification, which has the potential to impact SARS-CoV-2 genome maintenance.

Studying the intersection of nucleoside modifications and SARS-CoV-2 RNA-dependent RNA transcription using an in vitro reconstituted system.

There is growing recognition that viral RNA genomes possess enzymatically incorporated modified nucleosides. These small chemical changes are analogous to epigenomic modifications in DNA and have the potential to be similarly important modulators of viral transcription and evolution. However, the molecular level consequences of individual sites of modification remain to be broadly explored. Here we describe an in vitro assay to examine the impact of nucleoside modifications on the rate and fidelity of SARS-CoV-2 RNA transcription. Establishing the role of modified nucleotides in SARS-CoV-2 is of interest both for advancing fundamental knowledge of RNA modifications in viruses, and because modulating the modification-landscape of SARS-CoV-2 may represent a therapeutic strategy to interfere with viral RNA replication. Our approach can be used to assess the influence both of modifications present in a template RNA, as well nucleotide analog inhibitors. These methods provide a reproducible guide for generating active SARS-CoV-2 replication/transcription complexes capable of establishing how RNA modifications influence the pre-steady state rate constants of nucleotide addition by RNA-dependent RNA polymerases.

Methylated guanosine and uridine modifications in S. cerevisiae mRNAs modulate translation elongation.

Chemical modifications to protein encoding messenger RNAs (mRNAs) influence their localization, translation, and stability within cells. Over 15 different types of mRNA modifications have been observed by sequencing and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) approaches. While LC-MS/MS is arguably the most essential tool available for studying analogous protein post-translational modifications, the high-throughput discovery and quantitative characterization of mRNA modifications by LC-MS/MS has been hampered by the difficulty of obtaining sufficient quantities of pure mRNA and limited sensitivities for modified nucleosides. We have overcome these challenges by improving the mRNA purification and LC-MS/MS pipelines. The methodologies we developed result in no detectable non-coding RNA modifications signals in our purified mRNA samples, quantify 50 ribonucleosides in a single analysis, and provide the lowest limit of detection reported for ribonucleoside modification LC-MS/MS analyses. These advancements enabled the detection and quantification of 13 S. cerevisiae mRNA ribonucleoside modifications and reveal the presence of four new S. cerevisiae mRNA modifications at low to moderate levels (1-methyguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, and 5-methyluridine). We identified four enzymes that incorporate these modifications into S. cerevisiae mRNAs (Trm10, Trm11, Trm1, and Trm2, respectively), though our results suggest that guanosine and uridine nucleobases are also non-enzymatically methylated at low levels. Regardless of whether they are incorporated in a programmed manner or as the result of RNA damage, we reasoned that the ribosome will encounter the modifications that we detect in cells. To evaluate this possibility, we used a reconstituted translation system to investigate the consequences of modifications on translation elongation. Our findings demonstrate that the introduction of 1-methyguanosine, N2-methylguanosine and 5-methyluridine into mRNA codons impedes amino acid addition in a position dependent manner. This work expands the repertoire of nucleoside modifications that the ribosome must decode in S. cerevisiae. Additionally, it highlights the challenge of predicting the effect of discrete modified mRNA sites on translation de novo because individual modifications influence translation differently depending on mRNA sequence context.

Conserved 5-methyluridine tRNA modification modulates ribosome translocation.

While the centrality of post-transcriptional modifications to RNA biology has long been acknowledged, the function of the vast majority of modified sites remains to be discovered. Illustrative of this, there is not yet a discrete biological role assigned for one the most highly conserved modifications, 5-methyluridine at position 54 in tRNAs (m 5 U54). Here, we uncover contributions of m 5 U54 to both tRNA maturation and protein synthesis. Our mass spectrometry analyses demonstrate that cells lacking the enzyme that installs m 5 U in the T-loop (TrmA in E. coli , Trm2 in S. cerevisiae ) exhibit altered tRNA modifications patterns. Furthermore, m 5 U54 deficient tRNAs are desensitized to small molecules that prevent translocation in vitro. This finding is consistent with our observations that, relative to wild-type cells, trm2 Δ cell growth and transcriptome-wide gene expression are less perturbed by translocation inhibitors. Together our data suggest a model in which m 5 U54 acts as an important modulator of tRNA maturation and translocation of the ribosome during protein synthesis.

The perinatal transition of the circulating metabolome in a nonhuman primate.

INTRODUCTION: The fetal-to-neonatal transition is one of the most complex processes in biological existence; much is unknown about this transition on the molecular and biochemical level. Based on growing metabolomics literature, we hypothesize that metabolomic analysis will reveal the key biochemical intermediates that change during the birth transition. RESULTS: Using two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC × GC-TOFMS), we identified 100 metabolites that changed during this transition. Of these 100 metabolites, 23 demonstrated significant change during the first 72 h. Of note, four intermediates of the tricarboxylic acid (TCA) cycle were identified (α-ketoglutaric acid, fumaric acid, malic acid, and succinyl-CoA), demonstrating a consistent rate of rise during the study. This may signify the transition of the neonate from a hypoxic in utero environment to an oxygen-rich environment. Important signaling molecules were also identified, including myo-inositol and glutamic acid. DISCUSSION: GC × GC-TOFMS was able to identify important metabolites associated with metabolism and signaling. These data can be used as a baseline for normal birth transition, which may aid in future perinatal research investigations. METHODS: Late-preterm Macaca nemestrina were delivered by hysterotomy, with plasma drawn from the cord blood and after birth at eight additional time points to 72 h of age.

Sequence-specific capture and concentration of viral RNA by type III CRISPR system enhances diagnostic.

Type-III CRISPR-Cas systems have recently been adopted for sequence-specific detection of SARS-CoV-2. Here, we repurpose the type III-A CRISPR complex from Thermus thermophilus (TtCsm) for programmable capture and concentration of specific RNAs from complex mixtures. The target bound TtCsm complex generates two cyclic oligoadenylates (i.e., cA3 and cA4) that allosterically activate ancillary nucleases. We show that both Can1 and Can2 nucleases cleave single-stranded RNA, single-stranded DNA, and double-stranded DNA in the presence of cA4. We integrate the Can2 nuclease with type III-A RNA capture and concentration for direct detection of SARS-CoV-2 RNA in nasopharyngeal swabs with 15 fM sensitivity. Collectively, this work demonstrates how type-III CRISPR-based RNA capture and concentration simultaneously increases sensitivity, limits time to result, lowers cost of the assay, eliminates solvents used for RNA extraction, and reduces sample handling.

Sequence-specific capture and concentration of viral RNA by type III CRISPR system enhances diagnostic.

Type-III CRISPR-Cas systems have recently been adopted for sequence-specific detection of SARS-CoV-2. Here, we make two major advances that simultaneously limit sample handling and significantly enhance the sensitivity of SARS-CoV-2 RNA detection directly from patient samples. First, we repurpose the type III-A CRISPR complex from Thermus thermophilus (TtCsm) for programmable capture and concentration of specific RNAs from complex mixtures. The target bound TtCsm complex primarily generates two cyclic oligoadenylates (i.e., cA3 and cA4) that allosterically activate ancillary nucleases. To improve sensitivity of the diagnostic, we identify and test several ancillary nucleases (i.e., Can1, Can2, and NucC). We show that Can1 and Can2 are activated by both cA3 and cA4, and that different activators trigger changes in the substrate specificity of these nucleases. Finally, we integrate the type III-A CRISPR RNA-guided capture technique with the Can2 nuclease for 90 fM (5x104 copies/ul) detection of SARS-CoV-2 RNA directly from nasopharyngeal swab samples.

Parkinson-dementia complex and development of a new stable isotope dilution assay for BMAA detection in tissue.

Beta-methylamino-L-alanine (BMAA) has been proposed as a global contributor to neurodegenerative diseases, including Parkinson-dementia complex (PDC) of Guam and Alzheimer's disease (AD). The literature on the effects of BMAA is conflicting with some but not all in vitro data supporting a neurotoxic action, and experimental animal data failing to replicate the pattern of neurodegeneration of these human diseases, even at very high exposures. Recently, BMAA has been reported in human brain from individuals afflicted with PDC or AD. Some of the BMAA in human tissue reportedly is freely extractable (free) while some is protein-associated and liberated by techniques that hydrolyze the peptide bond. The latter is especially intriguing since BMAA is a non-proteinogenic amino acid that has no known tRNA. We attempted to replicate these findings with techniques similar to those used by others; despite more than adequate sensitivity, we were unable to detect free BMAA. Recently, using a novel stable isotope dilution assay, we again were unable to detect free or protein-associated BMAA in human cerebrum. Here we review the development of our new assay for tissue detection of BMAA and show that we are able to detect free BMAA in liver but not cerebrum, nor do we detect any protein-associated BMAA in mice fed this amino acid. These studies demonstrate the importance of a sensitive and specific assay for tissue BMAA and seriously challenge the proposal that BMAA is accumulating in human brain.

Rethinking cycad metabolite research.

Cycads are among the most ancient of extant Spermatophytes, and are known for their numerous pharmacologically active compounds. One compound in particular, ß-methylamino-L-alanine (BMAA), has been implicated as the cause of amyotrophic lateral sclerosis/Parkinson dementia complex (ALS/PDC) on Guam. Previous studies allege that BMAA is produced exclusively by cyanobacteria, and is transferred to cycads through the symbiotic relationship between these cyanobacteria and the roots of cycads. We recently published data showing that Cycas micronesica seedlings grown without endophytic cyanobacteria do in fact increase in BMAA, invalidating the foundation of the BMAA hypothesis. We use this example to suggest that the frenzy centered on BMAA and other single putative toxins has hindered progress. The long list of cycad-specific compounds may have important roles in signaling or communication, but these possibilities have been neglected during decades of attempts to force single metabolites into a supposed anti-herbivory function. We propose that an unbiased, comprehensive approach may be a more appropriate means of proceeding with cycad biochemistry research.

Application of comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry method to identify potential biomarkers of perinatal asphyxia in a non-human primate model.

Perinatal asphyxia is a leading cause of brain injury in infants, occurring in 2-4 per 1000 live births. The clinical response to asphyxia is variable and difficult to predict with current diagnostic tests. Reliable biomarkers are needed to help predict the timing and severity of asphyxia, as well as response to treatment. Two-dimensional gas chromatography-time-of-flight-mass spectrometry (GC×GC-TOFMS) was used herein, in conjunction with chemometric data analysis approaches for metabolomic analysis in order to identify significant metabolites affected by birth asphyxia. Blood was drawn before and after 15 or 18 min of cord occlusion in a Macaca nemestrina model of perinatal asphyxia. Postnatal samples were drawn at 5 min of age (n=20 subjects). Metabolomic profiles of asphyxiated animals were compared to four controls delivered at comparable gestational age. Fifty metabolites with the greatest change pre- to post-asphyxia were identified and quantified. The metabolic profile of post-asphyxia samples showed marked variability compared to the pre-asphyxia samples. Fifteen of the 50 metabolites showed significant elevation in response to asphyxia, ten of which remained significant upon comparison to the control animals. This metabolomic analysis confirmed lactate and creatinine as markers of asphyxia and discovered new metabolites including succinic acid and malate (intermediates in the Krebs cycle) and arachidonic acid (a brain fatty acid and inflammatory marker) as potential biomarkers. GC×GC-TOFMS coupled with chemometric data analysis are useful tools to identify acute biomarkers of brain injury. Further study is needed to correlate these metabolites with severity of disease, and response to treatment.

Cycas micronesica (Cycadales) plants devoid of endophytic cyanobacteria increase in beta-methylamino-L-alanine.

Cycads are among the most ancient of extant Spermatophytes, and are known for their pharmacologically active compounds. beta-methylamino-l-alanine (BMAA) is one metabolite that been implicated as causal of human neurodegenerative diseases in Guam. We grew Cycas micronesica seedlings without endophytic cyanobacteria symbiosis, and quantified initial and ending BMAA in various plant tissues. BMAA increased 79% during nine months of seedling growth, and root tissue contained 75% of the ultimate BMAA pool. Endophytic cyanobacteria symbionts were not the source of BMAA increase in these seedlings, which contradicts previously reported claims that biosynthesis of this toxin by cyanobacteria initiates its accumulation in the Guam environment. The preferential loading of root tissue with BMAA does not support earlier reports that this toxin serves a defensive role against herbivory of leaf or seed tissues. The long history of conflicting results in Guam's cycad toxin research continues, and recent developments underscore the sense of urgency in continued research as this endangered cycad population approaches extirpation from the island.

Development and application of a comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry method for the analysis of L-beta-methylamino-alanine in human tissue.

L-Beta-methylamino-alanine (BMAA) has been proposed as a worldwide contributor to neurodegenerative diseases, including Parkinson dementia complex (PDC) of Guam and Alzheimer's disease (AD). Recent conflicting reports of the presence of this amino acid in human brain from patients affected by these diseases have made it necessary to develop methods that provide unambiguous detection in complex samples. Comprehensive two-dimensional gas chromatography coupled with time-of-flight-mass-spectrometry analysis (GCxGC-TOFMS) followed by a targeted Parallel Factor Analysis (PARAFAC) deconvolution method has been used recently in metabolomic investigations to separate, identify, and quantify components of complex biological specimens. We have extended and applied this methodology to the toxicological problem of detecting BMAA in extracts of brain tissue. Our results show that BMAA can be isolated from closely eluting compounds and detected in trace amounts in extracts of brain tissue spiked with low levels of this analyte, ranging from 2.5ppb to 50ppb, with a limit of detection (LOD) of 0.7ppb. This new method was sufficiently sensitive to detect BMAA in cerebral extracts of mice fed BMAA. This optimized approach was then applied to analyze tissue from humans; however, no BMAA was detected in the brain extracts from controls or patients with PDC or AD. Our results demonstrate the application of multidimensional chromatography-mass spectrometry methods and computational deconvolution analysis to the problem of detecting trace amounts of a potential toxin in brain extracts from mice and humans.