Domain consolidation in Bacterial 50S assembly revealed by Anti-Sense Oligonucleotide Probing.

Investigating the intricate and rapid folding kinetics of large RNA-protein complexes (RNPs), like the bacterial ribosome, remains a formidable challenge in structural biology. Previous genetic approaches to probe assembly have focused on modulating the expression of either r-proteins or assembly factors. Here, anti-sense oligonucleotides (ASOs) were used to disrupt native RNA/RNA and RNA/protein interactions, in order to generate novel folding intermediates. In an in vitro co-transcriptional assembly assay, 8 assembly inhibitor ASOs were identified. Using cryo-electron microscopy, 38 new intermediate structures were determined resulting from the specific inhibitions. In particular a novel intermediate class provided compelling evidence of independent rRNA domain folding before proper interdomain docking. Three PNAs targeting domain-I of 23S- rRNA further subdivided the previously identified assembly core into smaller blocks representing the earliest steps in assembly. The resulting assembly graph reveals template-directed RNA foldon docking and domain consolidation, which provides a hierarchical view of the RNP assembly process.

Complete list of canonical post-transcriptional modifications in the Bacillus subtilis ribosome and their link to RbgA driven large subunit assembly.

Ribosomal RNA modifications in prokaryotes have been sporadically studied, but there is a lack of a comprehensive picture of modification sites across bacterial phylogeny. B. subtilis is a preeminent model organism for gram-positive bacteria, with a well-annotated and editable genome, convenient for fundamental studies and industrial use. Yet remarkably, there has been no complete characterization of its rRNA modification inventory. By expanding modern MS tools for the discovery of RNA modifications, we found a total of 25 modification sites in 16S and 23S rRNA of B. subtilis, including the chemical identity of the modified nucleosides and their precise sequence location. Furthermore, by perturbing large subunit biogenesis using depletion of an essential factor RbgA and measuring the completion of 23S modifications in the accumulated intermediate, we provide a first look at the order of modification steps during the late stages of assembly in B. subtilis . While our work expands the knowledge of bacterial rRNA modification patterns, adding B. subtilis to the list of fully annotated species after E. coli and T. thermophilus, in a broader context, it provides the experimental framework for discovery and functional profiling of rRNA modifications to ultimately elucidate their role in ribosome biogenesis and translation.

Assembly of the Bacterial Ribosome with Circularly Permuted rRNA.

Co-transcriptional assembly is an integral feature of the formation of RNA-protein complexes that mediate translation. For ribosome synthesis, prior studies have indicated that the strict order of transcription of rRNA domains may not be obligatory during bacterial ribosome biogenesis, since a series of circularly permuted rRNAs are viable. In this work, we report the insights into assembly of the bacterial ribosome large subunit (LSU) based on cryo-EM density maps of intermediates that accumulate during in vitro ribosome synthesis using a set of circularly permuted (CiPer) rRNAs. The observed ensemble of twenty-three resolved ribosome large subunit intermediates reveals conserved assembly routes with an underlying hierarchy among cooperative assembly blocks. There are intricate interdependencies for the formation of key structural rRNA helices revealed from the circular permutation of rRNA. While the order of domain synthesis is not obligatory, the order of domain association does appear to proceed with a particular order, likely due to the strong evolutionary pressure on efficient ribosome synthesis. This work reinforces the robustness of the known assembly hierarchy of the bacterial large ribosomal subunit, and offers a coherent view of how efficient assembly of CiPer rRNAs can be understood in that context.

Assembly landscape for the bacterial large ribosomal subunit.

Assembly of ribosomes in bacteria is highly efficient, taking ~2-3 min, but this makes the abundance of assembly intermediates very low, which is a challenge for mechanistic understanding. Genetic perturbations of the assembly process create bottlenecks where intermediates accumulate, facilitating structural characterization. We use cryo-electron microscopy, with iterative subclassification to identify intermediates in the assembly of the 50S ribosomal subunit from E. coli. The analysis of the ensemble of intermediates that spans the entire biogenesis pathway for the 50 S subunit was facilitated by a dimensionality reduction and cluster picking approach using PCA-UMAP-HDBSCAN. The identity of the cooperative folding units in the RNA with associated proteins is revealed, and the hierarchy of these units reveals a complete assembly map for all RNA and protein components. The assembly generally proceeds co-transcriptionally, with some flexibility in the landscape to ensure efficiency for this central cellular process under a variety of growth conditions.

Near-physiological in vitro assembly of 50S ribosomes involves parallel pathways.

Understanding the assembly principles of biological macromolecular complexes remains a significant challenge, due to the complexity of the systems and the difficulties in developing experimental approaches. As a ribonucleoprotein complex, the ribosome serves as a model system for the profiling of macromolecular complex assembly. In this work, we report an ensemble of large ribosomal subunit intermediate structures that accumulate during synthesis in a near-physiological and co-transcriptional in vitro reconstitution system. Thirteen pre-50S intermediate maps covering the entire assembly process were resolved using cryo-EM single-particle analysis and heterogeneous subclassification. Segmentation of the set of density maps reveals that the 50S ribosome intermediates assemble based on fourteen cooperative assembly blocks, including the smallest assembly core reported to date, which is composed of a 600-nucleotide-long folded rRNA and three ribosomal proteins. The cooperative blocks assemble onto the assembly core following defined dependencies, revealing the parallel pathways at both early and late assembly stages of the 50S subunit.

Milankovitch theory and monsoon.

The widely accepted "Milankovitch theory" explains insolation-induced waxing and waning of the ice sheets and their effect on the global climate on orbital timescales. In the past half century, however, the theory has often come under scrutiny, especially regarding its "100-ka problem." Another drawback, but the one that has received less attention, is the "monsoon problem," which pertains to the exclusion of monsoon dynamics in classic Milankovitch theory even though the monsoon prevails over the vast low-latitude (∼30° N to ∼30° S) region that covers half of the Earth's surface and receives the bulk of solar radiation. In this review, we discuss the major issues with the current form of Milankovitch theory and the progress made at the research forefront. We suggest shifting the emphasis from the ultimate outcomes of the ice volume to the causal relationship between changes in northern high-latitude insolation and ice age termination events (or ice sheet melting rate) to help reconcile the classic "100-ka problem." We discuss the discrepancies associated with the characterization of monsoon dynamics, particularly the so-called "sea-land precession-phase paradox" and the "Chinese 100-ka problem." We suggest that many of these discrepancies are superficial and can be resolved by applying a holistic "monsoon system science" approach. Finally, we propose blending the conventional Kutzbach orbital monsoon hypothesis, which calls for summer insolation forcing of monsoons, with Milankovitch theory to formulate a combined "Milankovitch-Kutzbach hypothesis" that can potentially explain the dual nature of orbital hydrodynamics of the ice sheet and monsoon systems, as well as their interplays and respective relationships with the northern high-latitude insolation and inter-tropical insolation differential.

Coupled atmosphere-ice-ocean dynamics during Heinrich Stadial 2.

Our understanding of climate dynamics during millennial-scale events is incomplete, partially due to the lack of their precise phase analyses under various boundary conditions. Here we present nine speleothem oxygen-isotope records from mid-to-low-latitude monsoon regimes with sub-centennial age precision and multi-annual resolution, spanning the Heinrich Stadial 2 (HS2) - a millennial-scale event that occurred at the Last Glacial Maximum. Our data suggests that the Greenland and Antarctic ice-core chronologies require +320- and +400-year adjustments, respectively, supported by extant volcanic evidence and radiocarbon ages. Our chronological framework shows a synchronous HS2 onset globally. Our records precisely characterize a centennial-scale abrupt "tropical atmospheric seesaw" superimposed on the conventional "bipolar seesaw" at the beginning of HS2, implying a unique response/feedback from low-latitude hydroclimate. Together with our observation of an early South American monsoon shift at the HS2 termination, we suggest a more active role of low-latitude hydroclimate dynamics underlying millennial events than previously thought.

Protracted Indian monsoon droughts of the past millennium and their societal impacts.

Protracted droughts lasting years to decades constitute severe threats to human welfare across the Indian subcontinent. Such events are, however, rare during the instrumental period (ca. since 1871 CE). In contrast, the historic documentary evidence indicates the repeated occurrences of protracted droughts in the region during the preinstrumental period implying that either the instrumental observations underestimate the full spectrum of monsoon variability or the historic accounts overestimate the severity and duration of the past droughts. Here we present a temporally precise speleothem-based oxygen isotope reconstruction of the Indian summer monsoon precipitation variability from Mawmluh cave located in northeast India. Our data reveal that protracted droughts, embedded within multidecadal intervals of reduced monsoon rainfall, frequently occurred over the past millennium. These extreme events are in striking temporal synchrony with the historically documented droughts, famines, mass mortality events, and geopolitical changes in the Indian subcontinent. Our findings necessitate reconsideration of the region's current water resources, sustainability, and mitigation policies that discount the possibility of protracted droughts in the future.

Thermostable ionizable lipid-like nanoparticle (iLAND) for RNAi treatment of hyperlipidemia.

Small interfering RNA (siRNA) therapeutic is considered to be a promising modality for the treatment of hyperlipidemia. Establishment of a thermostable clinically applicable delivery system remains a most challenging issue for siRNA drug development. Here, a series of ionizable lipid-like materials were rationally designed; 4 panels of lipid formulations were fabricated and evaluated on the basis of four representative structures. The lead lipid (A1-D1-5) was stable at 40°C, and the optimized formulation (iLAND) showed dose and time dual-dependent gene silencing pattern with median effective dose of 0.18 mg/kg. In addition, potent and durable reduction of serum cholesterol and triglyceride were achieved by administering siRNAs targeting angiopoietin-like 3 or apolipoprotein C3 (APOC3) in high-fat diet-fed mice, db/db mice, and human APOC3 transgenic mice, respectively, accompanied by displaying ideal safety profiles. Therefore, siRNA@iLAND prepared with thermostable A1-D1-5 demonstrates substantial value for siRNA delivery, hyperlipidemia therapy, and prevention of subsequent metabolic diseases.

Collapse of the Liangzhu and other Neolithic cultures in the lower Yangtze region in response to climate change.

The Liangzhu culture in the Yangtze River Delta (YRD) was among the world's most advanced Neolithic cultures. Archeological evidence suggests that the Liangzhu ancient city was abandoned, and the culture collapsed at ~4300 years ago. Here, we present speleothem records from southeastern China in conjunction with other paleoclimatic and archeological data to show that the Liangzhu culture collapsed within a short and anomalously wet period between 4345 ± 32 and 4324 ± 30 years ago, supporting the hypothesis that the city was abandoned after large-scale flooding and inundation. We further show that the demise of Neolithic cultures in the YRD occurred within an extended period of aridity that started at ~4000 ± 45 years ago. We suggest that the major hydroclimatic changes between 4300 and 3000 years ago may have resulted from an increasing frequency of the El Niño­Southern Oscillation in the context of weakened Northern Hemisphere summer insolation.

Transcription and Reverse Transcription of an Expanded Genetic Alphabet In Vitro and in a Semisynthetic Organism.

Through the development of unnatural base pairs that are compatible with native DNA and RNA polymerases and the ribosome, we have expanded the genetic alphabet and enabled in vitro and in vivo production of proteins containing noncanonical amino acids. However, the absence of assays to characterize transcription has prevented the deconvolution of the contributions of transcription and translation to the reduced performance of some unnatural codons. Here we show that RNA containing the unnatural nucleotides is efficiently reverse transcribed into cDNA, and we develop an assay to measure the combined fidelity of transcription and reverse transcription. With this assay, we examine the performance of a wide variety of unnatural codons, both in vitro and in the in vivo environment of a semisynthetic organism. We find that transcription is generally efficient, decoding at the ribosome is generally more challenging, and, correspondingly, sequence-dependent translation efficiency is the origin of variable codon performance.

Timing and structure of the Younger Dryas event and its underlying climate dynamics.

The Younger Dryas (YD), arguably the most widely studied millennial-scale extreme climate event, was characterized by diverse hydroclimate shifts globally and severe cooling at high northern latitudes that abruptly punctuated the warming trend from the last glacial to the present interglacial. To date, a precise understanding of its trigger, propagation, and termination remains elusive. Here, we present speleothem oxygen-isotope data that, in concert with other proxy records, allow us to quantify the timing of the YD onset and termination at an unprecedented subcentennial temporal precision across the North Atlantic, Asian Monsoon-Westerlies, and South American Monsoon regions. Our analysis suggests that the onsets of YD in the North Atlantic (12,870 ± 30 B.P.) and the Asian Monsoon-Westerlies region are essentially synchronous within a few decades and lead the onset in Antarctica, implying a north-to-south climate signal propagation via both atmospheric (decadal-time scale) and oceanic (centennial-time scale) processes, similar to the Dansgaard-Oeschger events during the last glacial period. In contrast, the YD termination may have started first in Antarctica at ∼11,900 B.P., or perhaps even earlier in the western tropical Pacific, followed by the North Atlantic between ∼11,700 ± 40 and 11,610 ± 40 B.P. These observations suggest that the initial YD termination might have originated in the Southern Hemisphere and/or the tropical Pacific, indicating a Southern Hemisphere/tropics to North Atlantic-Asian Monsoon-Westerlies directionality of climatic recovery.

On the role of synthesized hydroxylated chalcones as dual functional amyloid-ß aggregation and ferroptosis inhibitors for potential treatment of Alzheimer's disease.

In addition to amyloid cascade hypothesis, ferroptosis - a recently identified cell death pathway associated with the accumulation of lipid hydroperoxides - was hypothesized as one of the main forms of cell death in Alzheimer's disease. Herein, a series of hydroxylated chalcones were designed and synthesized as dual-functional inhibitors to inhibit amyloid-ß peptide (Aß) aggregation as well as ferroptosis simultaneously. Thioflavin-T assay indicated trihydroxy chalcones inhibited Aß aggregation better. In human neuroblastoma SH-SY5Y cells, cytoprotective chalcones 14a-c with three hydroxyl substituents exhibited a significant neuroprotection against Aß1-42 aggregation induced toxicity. In addition, chalcones 14a-c were found to be good inhibitors of ferroptosis induced by either pharmacological inhibition of the hydroperoxide-detoxifying enzyme Gpx4 using (1S, 3R)-RSL4 or cystine/glutamate antiporter system Xc- inhibition by erastin through lipid peroxidation inhibition mechanism. Trihydroxy chalcone 14a was also able to completely subvert lipid peroxidation induced by Aß1-42 aggregation in SH-SY5Y cells indicating that they can reduce the neurotoxicity involved with oxidative stress. Compound 14a-c showed good ADMET properties and blood-brain barrier penetration in silico simulation software. From these data, a picture emerges wherein trihydroxy chalcones are potential candidates for the treatment of Alzheimer's disease by simultaneously inhibition of Aß1-42 aggregation and ferroptosis.