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Stimulated cells and cancer cells have widespread shortening of mRNA 3’-untranslated regions (3’UTRs) and switches to shorter mRNA isoforms due to usage of more proximal polyadenylation signals (PASs) in introns and last exons. U1 snRNP (U1), vertebrates’ most abundant non-coding (spliceosomal) small nuclear RNA, silences proximal PASs and its inhibition with antisense morpholino oligonucleotides (U1 AMO) triggers widespread premature transcription termination and mRNA shortening. Here we show that low U1 AMO doses increase cancer cells’ migration and invasion in vitro by up to 500%, whereas U1 over-expression has the opposite effect. In addition to 3’UTR length, numerous transcriptome changes that could contribute to this phenotype are observed, including alternative splicing, and mRNA expression levels of proto-oncogenes and tumor suppressors. These findings reveal an unexpected role for U1 homeostasis (available U1 relative to transcription) in oncogenic and activated cell states, and suggest U1 as a potential target for their modulation. U1 snRNP is a key regulator of mRNA biogenesis through its roles in splicing, and transcription and 3’-end processing. Here the authors show a tumor suppressor-like function of U1 snRNP using in vitro cell migration/invasion assays and transcriptome profiling.

The higher abundance of U1 among snRNPs is explained by the identification of an additional mode of assembly: RBP U1-70K bridges pre-U1 to SMN–Gemin2–Sm, thus establishing a Gemin5-independent assembly pathway. Despite equal snRNP stoichiometry in spliceosomes, U1 snRNP (U1) is typically the most abundant vertebrate snRNP. Mechanisms regulating U1 overabundance and snRNP repertoire are unknown. In Sm-core assembly, a key snRNP-biogenesis step mediated by the SMN complex, the snRNA-specific RNA-binding protein (RBP) Gemin5 delivers pre-snRNAs, which join SMN–Gemin2–recruited Sm proteins. We show that the human U1-specific RBP U1-70K can bridge pre-U1 to SMN–Gemin2–Sm, in a Gemin5-independent manner, thus establishing an additional and U1-exclusive Sm core–assembly pathway. U1-70K hijacks SMN–Gemin2–Sm, enhancing Sm-core assembly on U1s and inhibiting that on other snRNAs, thereby promoting U1 overabundance and regulating snRNP repertoire. SMN–Gemin2's ability to facilitate transactions between different RBPs and RNAs explains its multi-RBP valency and the myriad transcriptome perturbations associated with SMN deficiency in neurodegenerative spinal muscular atrophy. We propose that SMN–Gemin2 is a versatile hub for RNP exchange that functions broadly in RNA metabolism.

This thesis advances causal-inference methodology by shifting the question from what is the average effect of an intervention to how those effects arise. In Chapter 2, we develop a unified sensitivity analysis framework for heterogeneous treatment effects in observational studies. Building on Rosenbaum’s Γ -model, we extend and compare two leading approaches (HSR and submax) along with a hybrid submax+ method, to enable subgroup discovery, effect estimation, and sensitivity analysis on the same sample without inflating Type I error. Through extensive simulation and an application to Alzheimer’s Disease Neuroimaging Initiative data on sleep disturbances and cognitive decline, we evaluate statistical power and effect modifier detection, refine matching and inference procedures (including closed-testing and Γ -amplification), and release sensitivityhte, an R package that streamlines the end-to-end workflow for assessing the robustness of heterogeneous treatment-effect conclusions.In Chapter 3, we introduce CMAPS (Causal Mediation Analysis for Perturbation Screens), a semiparametric mediation framework tailored to single-cell CRISPR screens. CMAPS decomposes a perturbation’s total effect on gene expression into natural direct and indirect components mediated by a specified molecular intermediate. When applied to a genomescale Perturb-seq dataset in K562 cells, CMAPS reconstructs gene-specific pathways by which transcription factors propagate perturbation effects and highlights key mediators of GATA1 knockout. In a MultiPerturb-seq screen of BT16 cells, CMAPS further distinguishes promoter-mediated from enhancer-mediated regulation, revealing three discrete regulatory programs with potential therapeutic relevance.Together, these two chapters deliver tools for unraveling how causal effects arise: Chapter 2 equips researchers with sensitivity-analysis methods to assess the reliability of heterogeneous treatment effects in observational studies, while Chapter 3 provides a principled mediation framework for elucidating molecular pathways in single-cell perturbation data. By integrating advanced statistical techniques with practical software implementations, this thesis deepens our mechanistic insights in both observational studies and functional-genomic contexts.

Shq1 is a conserved protein required for the biogenesis of eukaryotic H/ACA ribonucleoproteins (RNPs), including human telomerase. We report the structure of the Shq1‐specific domain alone and in complex with H/ACA RNP proteins Cbf5, Nop10 and Gar1. The Shq1‐specific domain adopts a novel helical fold and primarily contacts the PUA domain and the otherwise disordered C‐terminal extension (CTE) of Cbf5. The structure shows that dyskeratosis congenita mutations found in the CTE of human Cbf5 likely interfere with Shq1 binding. However, most mutations in the PUA domain are not located at the Shq1‐binding surface and also have little effect on the yeast Cbf5–Shq1 interaction. Shq1 binds Cbf5 independently of the H/ACA RNP proteins Nop10, Gar1 and Nhp2 and the assembly factor Naf1, but shares an overlapping binding surface with H/ACA RNA. Shq1 point mutations that disrupt Cbf5 interaction suppress yeast growth particularly at elevated temperatures. Our results suggest that Shq1 functions as an assembly chaperone that protects the Cbf5 protein complexes from non‐specific RNA binding and aggregation before assembly of H/ACA RNA. Shq1 is required for the biogenesis of eukaryotic H/ACA ribonucleoproteins (RNPs) involved in telomere maintenance and pseudo‐uridylation of rRNA and snRNA. This study describes the crystal structure of Shq1 in complex with three H/ACA RNP proteins, suggesting that Shq1 acts as an assembly chaperone of immature RNPs.

Despite their equal stoichiometry in spliceosomes, U1 snRNP (U1) is typically the most abundant snRNP in vertebrates. What regulates U1 over-abundance and snRNP repertoire in general is unknown. Sm core assembly is a key step in snRNP biogenesis mediated by the SMN complex. All pre-snRNAs are delivered by the snRNA-specific RNA-binding protein (RBP) Gemin5 to join SMN-Gemin2-recruited Sm proteins. Here, we find that the U1-specific RBP U1-70K bridges pre-U1 to SMN-Gemin2-Sm, establishing an additional, Gemin5-independent Sm core assembly pathway. We show that U1-70K hijacks SMN-Gemin2-Sm, enhancing U1’s and inhibiting other snRNAs’ Sm core assembly, thereby promoting U1 over-abundance and regulating snRNP repertoire. Ubiquitous SMN-Gemin2’s surprising ability to facilitate transactions between different RBPs and RNAs explains its puzzling multi-RBP valency and myriad transcriptome perturbations associated with SMN’s deficiency in neurodegenerative spinal muscular atrophy. We propose that SMN-Gemin2 is a versatile RNP exchange that functions broadly in RNA metabolism.

The patho-mechanism of apolipoprotein variant, APOE4, the strongest genetic risk for late-onset Alzheimer's disease (AD) and longevity, remains unclear. APOE's neighboring gene, TOMM40 (mitochondria protein transport channel), is associated with brain trauma outcome and aging-related cognitive decline, however its role in AD APOE4-independently is controversial. We report that TOMM40 is prone to transcription readthrough into APOE that can generate spliced TOMM40-APOE mRNA chimera (termed T9A2) detected in human neurons and other cells and tissues. T9A2 translation tethers APOE (normal APOE3 or APOE4) to near-full-length TOM40 that is targeted to mitochondria. Importantly, T9A2-APOE3 boosts mitochondrial bioenergetic capacity and decreases oxidative stress significantly more than T9A2-APOE4 and APOE3, and lacking in APOE4. We describe detailed interactomes of these actors that may inform about the activities and roles in pathogenesis. T9A2 uncovers a new candidate pathway for mitochondria regulation and oxidative stress-protection that are impaired in APOE4 genotypes and could initiate neurodegeneration.

Elevated O3 concentrations pose a significant threat to human health and ecosystems, but little research has been performed on coastal wetlands near large cities. This study focuses on investigating the key factors affecting O3 formation in the ecologically sensitive Dongtan Wetland (Chongming District, Shanghai, China) area. By comparing the performance of O3 concentration prediction of multiple machine learning models, this study found that the random forest model achieved the highest accuracy (R2 = 0.9, RMSE = 11.5). Feature importance and structure mining showed that peroxyacetyl nitrate (PAN), nitrogen oxides (NOx), temperature, wind direction, and relative humidity were the main drivers of O3 formation. Specifically, PAN concentrations exceeding 0.1 ppb and temperatures above 3 °C were found to have a significant impact on O3 levels, especially in spring, summer, and autumn. Trajectory analysis showed that westward urban pollution and emissions transported from the ocean were the main factors in O3 formation in the area. This study highlights the need for targeted emission control strategies, especially for PAN precursors generated by ships and NOx generated by urban industries, providing important insights for improving air quality in ecologically sensitive coastal areas.

Water oxidation is the prerequisite for dioxygen evolution in natural or artificial photosynthesis. Although it has been demonstrated that multinuclear active sites are commonly necessary for water oxidation, as inspired by the natural oxygen-evolving centre CaMn 4 O 5 , a multinuclear manganese cluster, whether mononuclear manganese can also efficiently catalyse water oxidation has been a long-standing question. Herein, we found that a heterogeneous catalyst with mononuclear manganese embedded in nitrogen-doped graphene (Mn-NG) shows a turnover frequency as high as 214 s −1 for chemical water oxidation and an electrochemical overpotential as low as 337 mV at a current density of 10 mA cm −2 . Structural characterization and density functional theory calculations reveal that the high activity of Mn-NG can be attributed to the mononuclear manganese ion coordinated with four nitrogen atoms embedded in the graphene matrix. Nature’s oxygen-evolving complex of photosystem II is a multinuclear manganese cluster. Whether mononuclear manganese can also efficiently catalyse water oxidation has been a long-standing question. Now, Li and co-workers show that single atoms of manganese can be anchored on nitrogen-doped graphene to catalyse the oxygen evolution reaction. Credit: Water image Frankie Angel / Alamy Stock Photo.

Dual-metal catalysts with synergistic effect exhibit enormous potential for sustainable electrocatalytic applications and mechanism research. Compared with mono-metal-site catalysts, dual-metal-site catalysts exhibit higher efficiency for the oxygen evolution reaction (OER) due to reduced energy barrier of the process involving proton-coupled multi-electron transfer. Herein, we construct dual-metal Fe-Co sites coordinated with nitrogen in graphene (FeCo-NG), which exhibits high OER performance with onset overpotential of only 126 mV and Tafel slope of 120 mV·dec −1 , showing that the rate-determining step is controlled by the single-electron transfer step. Theoretical calculations reveal that the FeN 4 site exhibits lower OER overpotential than the CoN 4 site due to appropriate adsorption energy of OOH* on the former, while the O* adsorbed on the adjacent Co site could stabilize the OOH* on the FeN 4 site through hydrogen bond interaction.

Outer membrane vesicles (OMVs) are immunogenic self-adjuvanting vesicles produced by Gram-negative bacteria such as Pseudomonas aeruginosa and Yersinia pseudotuberculosis . While the effects of OMVs on different antigens immune stimulation are not clear. In this study, we constructed recombinant Yersinia pseudotuberculosis ΔlpxL strain,with pBlue-PcrV and pBlue-OprF/I , and then purified ΔlpxL rOMV PcrV (rOMVyp2P)and ΔlpxL rOMV OprF/I (rOMVyp2F) and analyzed its effect on immune response and protection against Pseudomonas aeruginosa PAO1 infection. The results showed that OMV assists in eliciting similar humoral immune responses to PcrV and OprF/I antigens. ΔlpxL rOMV PcrV and ΔlpxL rOMV OprF/I elicited Th1/Th2 balanced immune response, and higher IgM and IgA antibodies.However, there are differences in immune protection for the pulmonary. The survival rate of mice in ΔlpxL rOMV PcrV group was 20%, which was significantly better than that in ΔlpxL rOMV OprF/I group. ΔlpxL OMV PcrV is better cooperation for Pseudomonas immune protection in lung.