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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. 

Transcription factors associate with architectural proteins to regulate genome organization and three-dimensional gene regulation. 

Abstract Single-molecule tracking (SMT) allows the study of transcription factor (TF) dynamics in the nucleus, giving important information regarding the diffusion and binding behavior of these proteins in the nuclear environment. Dwell time distributions obtained by SMT for most TFs appear to follow bi-exponential behavior. This has been ascribed to two discrete populations of TFs—one non-specifically bound to chromatin and another specifically bound to target sites, as implied by decades of biochemical studies. However, emerging studies suggest alternate models for dwell-time distributions, indicating the existence of more than two populations of TFs (multi-exponential distribution), or even the absence of discrete states altogether (power-law distribution). Here, we present an analytical pipeline to evaluate which model best explains SMT data. We find that a broad spectrum of TFs (including glucocorticoid receptor, oestrogen receptor, FOXA1, CTCF) follow a power-law distribution of dwell-times, blurring the temporal line between non-specific and specific binding, suggesting that productive binding may involve longer binding events than previously believed. From these observations, we propose a continuum of affinities model to explain TF dynamics, that is consistent with complex interactions of TFs with multiple nuclear domains as well as binding and searching on the chromatin template. 

Among metal additive manufacturing technologies, additive friction stir deposition stands out for its ability to create freeform and fully-dense structures without melting and solidification. Here, we employ a comparative approach to investigate the process-microstructure linkages in additive friction stir deposition, utilizing two materials with distinct thermomechanical behavior—an Al-Mg-Si alloy and Cu—both of which are challenging to print using beam-based additive processes. The deposited Al-Mg-Si is shown to exhibit a relatively homogeneous microstructure with extensive subgrain formation and a strong shear texture, whereas the deposited Cu is characterized by a wide distribution of grain sizes and a weaker shear texture. We show evidence that the microstructure in Al-Mg-Si primarily evolves by continuous dynamic recrystallization, including geometric dynamic recrystallization and progressive lattice rotation, while the heterogeneous microstructure of Cu results from discontinuous recrystallization during both deposition and cooling. In Al-Mg-Si, the continuous recrystallization progresses with an increase of the applied strain, which correlates with the ratio between the tool rotation rate and travel velocity. Conversely, the microstructure evolution in Cu is found to be less dependent on , instead varying more with changes to . This difference originates from the absence of Cu rotation in the deposition zone, which reduces the influence of tool rotation on strain development. We attribute the distinct process-microstructure linkages and the underlying mechanisms between Al-Mg-Si and Cu to their differences in intrinsic thermomechanical properties and interactions with the tool head. 

Additive friction stir deposition (AFSD) is an emerging solid-state metal additive manufacturing technology renowned for strong interface adhesion and isotropic mechanical properties. This is postulated to result from the material flow phenomena near the interface, but experimental corroboration has remained absent. Here, we seek to understand the interface formed in AFSD via morphological and microstructural investigation, wherein the non-planar interfacial morphology is characterized on the track-scale (centimeter scale) using X-ray computed tomography and the material deformation history is explored by microstructure mapping at the interfacial regions. X-ray computed tomography reveals unique 3D features at the interface with significant macroscopic material mixing. In the out-of-plane direction, the deposited material inserts below the initial substrate surface in the feed-rod zone, while the substrate surface surges upwards in the tool protrusion-affected zone. Complex 3D structures like fins and serrations form on the advancing side, leading to structural interlocking; on the retreating side, the interface manifests as a smooth sloped surface. Microstructure mapping reveals a uniform thermomechanical history for the deposited material, which develops a homogeneous, almost fully recrystallized microstructure. The substrate surface develops partially recrystallized microstructures that are location-dependent; more intra-granular orientation gradients are found in the regions further away from the centerline of the deposition track. From these observations, we discuss the mechanisms for interfacial material flow and interface morphology formation during AFSD.