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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 inEscherichia coli, Trm2 inSaccharomyces 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,trm2Δcell 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. 

Remote reconnaissance missions are promising solutions for the assessment of earthquake-induced structural damage and cascading geological hazards. Space-borne remote sensing can complement in-field missions when safety and accessibility concerns limit post-earthquake operations on the ground. However, the implementation of remote sensing techniques in post-disaster missions is limited by the lack of methods that combine different techniques and integrate them with field survey data. This paper presents a new approach for rapid post-earthquake building damage assessment and landslide mapping, based on Synthetic Aperture Radar (SAR) data. The proposed texture-based building damage classification approach exploits very high resolution post-earthquake SAR data integrated with building survey data. For landslide mapping, a backscatter intensity-based landslide detection approach, which also includes the separation between landslides and flooded areas, is combined with optical-based manual inventories. The approach was implemented during the joint Structural Extreme Event Reconnaissance, GeoHazards International and Earthquake Engineering Field Investigation Team mission that followed the 2021 Haiti Earthquake and Tropical Cyclone Grace. 

Abstract Transfer RNAs (tRNAs) contain dozens of chemical modifications. These modifications are critical for maintaining tRNA tertiary structure and optimizing protein synthesis. Here we advance the use of Nanopore direct RNA-sequencing (DRS) to investigate the synergy between modifications that are known to stabilize tRNA structure. We sequenced the 42 cytosolic tRNA isoacceptors from wild-type yeast and five tRNA-modifying enzyme knockout mutants. These data permitted comprehensive analysis of three neighboring and conserved modifications in T-loops: 5-methyluridine (m5U54), pseudouridine (Ψ55), and 1-methyladenosine (m1A58). Our results were validated using direct measurements of chemical modifications by mass spectrometry. We observed concerted T-loop modification circuits—the potent influence of Ψ55 for subsequent m1A58 modification on more tRNA isoacceptors than previously observed. Growing cells under nutrient depleted conditions also revealed a novel condition-specific increase in m1A58 modification on some tRNAs. A global and isoacceptor-specific classification strategy was developed to predict the status of T-loop modifications from a user-input tRNA DRS dataset, applicable to other conditions and tRNAs in other organisms. These advancements demonstrate how orthogonal technologies combined with genetics enable precise detection of modification landscapes of individual, full-length tRNAs, at transcriptome-scale. 

Among RNAs, transfer RNAs (tRNAs) contain the widest variety of abundant post-transcriptional chemical modifications. These modifications are crucial for tRNAs to participate in protein synthesis, promoting proper tRNA structure and aminoacylation, facilitating anticodon:codon recognition, and ensuring the reading frame maintenance of the ribosome. While tRNA modifications were long thought to be stoichiometric, it is becoming increasingly apparent that these modifications can change dynamically in response to the cellular environment. The ability to broadly characterize the fluctuating tRNA modification landscape will be essential for establishing the molecular level contributions of individual sites of tRNA modification. The locations of modifications within individual tRNA sequences can be mapped using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). In this approach, a single tRNA species is purified, treated with ribonucleases and the resulting single-stranded RNA products are subject to LC-MS/MS analysis. The application of LC-MS/MS to study tRNAs is limited by the necessity of analyzing one tRNA at a time because the digestion of total tRNA mixtures by commercially available ribonucleases produces many short digestion products unable to be uniquely mapped back to a single site within a tRNA. We overcame these limitations by taking advantage of the highly structured nature of tRNAs to prevent the full digestion by single-stranded RNA specific ribonucleases. Folding total tRNA prior to digestion allowed us to sequence S. cerevisiae tRNAs with up to 97% sequence coverage for individual tRNA species by LC-MS/MS. This method presents a robust avenue for directly detecting the distribution of modifications in total tRNAs. 

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 (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 inE. coli, Trm2 inS. cerevisiae) exhibit altered tRNA modifications patterns. Furthermore, m⁵U54 deficient tRNAs are desensitized to small molecules that prevent translocationin 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. 

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, N 2-methylguanosine, N 2, N 2-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, N 2-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. 

Indigenous Peoples across the Arctic have adapted to environmental change since time immemorial, yet recent climate change has imposed unprecedented and abrupt changes that affect the land and sea upon which communities rely. Co-created community-based observing programs offer an opportunity to harness the holistic breadth of knowledge in communities with the goal of tracking Arctic change while simultaneously supporting community priorities and local-scale needs. The Alaska Arctic Observatory and Knowledge Hub (AAOKH) is a network of Iñupiaq observers from northern Alaska coastal communities working in partnership with academic researchers. Here, we describe five core functions that have emerged through AAOKH, which include tracking long-term environmental changes; communicating Indigenous-led observations of the environment and their meaning; place-based and culturally relevant education; enabling scientific and Indigenous Knowledge exchange; and supporting community-led responses to environmental change. We outline and discuss specific actions and opportunities that have been used to increase knowledge exchange of AAOKH observations, make space for the next generation of Indigenous scholars, and create locally relevant data products and syntheses that can inform resource management and community planning. We also discuss our ongoing efforts to increasingly shift toward a knowledge coproduction framework as we plan to sustain AAOKH into the future.