Zero-shot prediction of mutation effects with multimodal deep representation learning guides protein engineering.

Mutations in amino acid sequences can provoke changes in protein function. Accurate and unsupervised prediction of mutation effects is critical in biotechnology and biomedicine, but remains a fundamental challenge. To resolve this challenge, here we present Protein Mutational Effect Predictor (ProMEP), a general and multiple sequence alignment-free method that enables zero-shot prediction of mutation effects. A multimodal deep representation learning model embedded in ProMEP was developed to comprehensively learn both sequence and structure contexts from ~160 million proteins. ProMEP achieves state-of-the-art performance in mutational effect prediction and accomplishes a tremendous improvement in speed, enabling efficient and intelligent protein engineering. Specifically, ProMEP accurately forecasts mutational consequences on the gene-editing enzymes TnpB and TadA, and successfully guides the development of high-performance gene-editing tools with their engineered variants. The gene-editing efficiency of a 5-site mutant of TnpB reaches up to 74.04% (vs 24.66% for the wild type); and the base editing tool developed on the basis of a TadA 15-site mutant (in addition to the A106V/D108N double mutation that renders deoxyadenosine deaminase activity to TadA) exhibits an A-to-G conversion frequency of up to 77.27% (vs 69.80% for ABE8e, a previous TadA-based adenine base editor) with significantly reduced bystander and off-target effects compared to ABE8e. ProMEP not only showcases superior performance in predicting mutational effects on proteins but also demonstrates a great capability to guide protein engineering. Therefore, ProMEP enables efficient exploration of the gigantic protein space and facilitates practical design of proteins, thereby advancing studies in biomedicine and synthetic biology.

Liver Fibrosis Amelioration by Macrophage-Biomimetic Polydopamine Nanoparticles via Synergistically Alleviating Inflammation and Scavenging ROS.

The progression of liver fibrosis is determined by the interaction of damaged hepatocytes, active hepatic stellate cells, and macrophages, contributing to the development of oxidative stress and inflammatory environments within the liver. Unfortunately, the current pharmacological treatment for liver fibrosis is limited by its inability to regulate inflammation and oxidative stress concurrently. In this study, we developed a cell membrane biomaterial for the treatment of liver fibrosis, which we designated as PM. PM is a biomimetic nanomaterial constructed by encapsulating polydopamine (PDA) with a macrophage membrane (MM). It is hypothesized that PM nanoparticles (NPs) can successfully target the site of inflammation, simultaneously inhibit inflammation, and scavenge reactive oxygen species (ROS). In vitro experiments demonstrated that PM NPs exhibited strong antioxidant properties and the ability to neutralize pro-inflammatory cytokines (TNF-α, IL-6, and IL-1ß). Moreover, the capacity of PM NPs to safeguard cells from oxidative stress and their anti-inflammatory efficacy in an inflammatory model were validated in subsequent cellular experiments. Additionally, PM NPs exhibited a high biocompatibility. In a mouse model of hepatic fibrosis, PM NPs were observed to aggregate efficiently in the fibrotic liver, displaying excellent antioxidant and anti-inflammatory properties. Notably, PM NPs exhibited superior targeting, anti-inflammatory, and ROS scavenging abilities in inflamed tissues compared to MM, PDA, or erythrocyte membrane-encapsulated PDA. Under the synergistic effect of anti-inflammation and antioxidant, PM NPs produced significant therapeutic effects on liver fibrosis in mice. In conclusion, the synergistic alleviation of inflammation and ROS scavenging by this specially designed nanomaterial, PM NPs, provides valuable insights for the treatment of liver fibrosis and other inflammatory- or oxidative stress-related diseases.

Maternal KLF17 controls zygotic genome activation by acting as a messenger for RNA Pol II recruitment in mouse embryos.

Initiation of timely and sufficient zygotic genome activation (ZGA) is crucial for the beginning of life, yet our knowledge of transcription factors (TFs) contributing to ZGA remains limited. Here, we screened the proteome of early mouse embryos after cycloheximide (CHX) treatment and identified maternally derived KLF17 as a potential TF for ZGA genes. Using a conditional knockout (cKO) mouse model, we further investigated the role of maternal KLF17 and found that it promotes embryonic development and full fertility. Mechanistically, KLF17 preferentially binds to promoters and recruits RNA polymerase II (RNA Pol II) in early 2-cell embryos, facilitating the expression of major ZGA genes. Maternal Klf17 knockout resulted in a downregulation of 9% of ZGA genes and aberrant RNA Pol II pre-configuration, which could be partially rescued by introducing exogenous KLF17. Overall, our study provides a strategy for screening essential ZGA factors and identifies KLF17 as a crucial TF in this process.

Near-Infrared Fluorescence Imaging of miRNA Using a Transmembrane Polypeptide-Based Genetic Reporter.

MicroRNAs (miRNAs) are small noncoding RNAs that play important regulatory roles in multiple biological processes. Many miRNAs exhibit unique expression patterns and are considered as theranostic biomarkers in a variety of human diseases. A reporter system that is capable of imaging miRNA in vivo is crucial for investigating miRNA biology. In the present study, an organic anion-transporting polypeptide 1B3 (OATP1B3)-based genetic switch system is designed and optimized to achieve near-infrared fluorescent imaging of miRNA by the uptake of indocyanine green (ICG) dye. The reporter system, named miR-ON-OB3, is shown to be efficient to regulate the expression of OATP1B3 in mammalian cells. Notably, the results indicate that the system is of high sensitivity for near-infrared fluorescence imaging of both exogenous and endogenous miRNA in mammalian cells. Moreover, the system is proved to be functional for real-time near-infrared fluorescence imaging of miRNA in living mice. This study establishes a novel genetic encoded reporter for near-infrared fluorescence imaging of miRNA, which may provide a potential tool for in vivo imaging of miRNA in clinical applications due to the clinical availability of ICG.

Nuezhenoside G13 from Osmanthus fragrans fruit ameliorates Concanavalin A-induced autoimmune hepatitis by regulating the NF-κB/MAPK pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Osmanthus fragrans fruit (OFF) exhibits hepatoprotective function, and it is consumed as food and used in traditional medicine in China. Nuezhenoside G13 (G13) is present in the highest levels in OFF. Autoimmune hepatitis (AIH) is a manifestation of liver disease and seriously endangers health. However, it remains unclear whether G13 affects AIH. AIM OF THE STUDY: To clarify the effect of G13 on AIH and its exact underlying mechanism from a new perspective. MATERIALS AND METHODS: We used a Concanavalin A-induced AIH mouse model and lipopolysaccharide-treated Raw264.7 cells to quantify serum biochemical indicators and confirm whether G13 exhibited protective effects in the AIH mice. Furthermore, we evaluated the effect of G13 via hematoxylin and eosin and immunohistochemical staining. We used enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction to quantify the inflammatory factors. We confirmed that G13 inhibited apoptosis via terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Molecular docking, immunofluorescence, and western blotting experiments of G13 and key proteins of the NF-κB/MAPK pathway revealed that G13 alleviated inflammation. In addition, Cell Counting Kit-8, ELISA, NO detection, and western blotting assays were performed. Finally, we used an inhibitor of the p38 MAPK to verify that G13 reduced inflammation through the NF-κB/MAPK pathway in Raw264.7 cells. RESULTS: The in vivo experiments revealed that G13 improved oxidative stress and apoptosis. In addition, G13 decreased the expression levels of CD4+, CD8+, F4/80+, and Ly6G and the secretion of inflammatory factors. Interestingly, G13 reduced the phosphorylation levels of IκBα, NF-κB, JNK, ERK1/2, and p38. Additionally, the in vitro experiments revealed that G13 alleviated inflammation through the NF-κB/MAPK pathway in lipopolysaccharide-treated Raw264.7 cells. Furthermore, molecular docking demonstrated that the binding fraction of G13 with these proteins was high. CONCLUSION: G13 suppressed oxidative stress, apoptosis, and inflammation in a Concanavalin A-induced AIH mouse model. Furthermore, G13 exerted its effect through the NF-κB/MAPK pathway.

A DddA ortholog-based and transactivator-assisted nuclear and mitochondrial cytosine base editors with expanded target compatibility.

Bacterial double-stranded DNA (dsDNA) cytosine deaminase DddAtox-derived cytosine base editor (DdCBE) and its evolved variant, DddA11, guided by transcription-activator-like effector (TALE) proteins, enable mitochondrial DNA (mtDNA) editing at TC or HC (H = A, C, or T) sequence contexts, while it remains relatively unattainable for GC targets. Here, we identified a dsDNA deaminase originated from a Roseburia intestinalis interbacterial toxin (riDddAtox) and generated CRISPR-mediated nuclear DdCBEs (crDdCBEs) and mitochondrial CBEs (mitoCBEs) using split riDddAtox, which catalyzed C-to-T editing at both HC and GC targets in nuclear and mitochondrial genes. Moreover, transactivator (VP64, P65, or Rta) fusion to the tail of DddAtox- or riDddAtox-mediated crDdCBEs and mitoCBEs substantially improved nuclear and mtDNA editing efficiencies by up to 3.5- and 1.7-fold, respectively. We also used riDddAtox-based and Rta-assisted mitoCBE to efficiently stimulate disease-associated mtDNA mutations in cultured cells and in mouse embryos with conversion frequencies of up to 58% at non-TC targets.

A dual-regulation inducible switch system for microRNA detection and cell type-specific gene activation.

Rationale: MicroRNAs (miRNAs) play key roles in multiple biological processes, many of which exhibit distinct cell type-specific expression patterns. A miRNA-inducible expression system can be adapted as a signal-on reporter for detecting miRNA activity or as a cell type-specific gene activation tool. However, due to the inhibitory properties of miRNAs on gene expression, few miRNA-inducible expression systems are available, and the available systems are only transcriptional or post-transcriptional regulatory system with obvious leaky expression. Methods: To address this limitation, a miRNA-inducible expression system that can tightly control target gene expression is desirable. Here, by taking advantage of an enhanced LacI repression system and the translational repressor L7Ae, a miRNA-inducible dual transcriptional-translational switch system was designed called the miR-ON-D system. Luciferase activity assay, western blotting, CCK-8 assay and flow cytometry analysis were performed to characterize and validate this system. Results: The results demonstrated that leakage expression was strongly suppressed in the miR-ON-D system. It was also validated that the miR-ON-D system could be used to detect exogenous and endogenous miRNAs in mammalian cells. Moreover, it was shown that the miR-ON-D system could be triggered by cell type-specific miRNAs to regulate the expression of biologically relevant proteins (e.g., p21 and Bax) to achieve cell type-specific reprogramming. Conclusion: This study established a tight miRNA-inducible expression switch system for miRNA detection and cell type-specific gene activation.

Engineering Synthetic CopT/A-Based Genetic Biosensors for miRNA Imaging and Functional Gene Regulation.

Synthetic genetic biosensors that can operate at the transcriptional and translation levels have been widely applied in the control of cellular behaviors and functions. However, the regulation of genetic circuits is often accompanied by the introduction of exogenous substances or the endogenous generation of inhibitory products, which would bring uncontrollable hazards to biological safety and reduce the efficiency of the system. Here, we described a miRNA-responsive CopT-CopA (miCop) genetic biosensor system to realize real-time monitoring of the intracellular expression of miRNA-124a during neurogenesis or miRNA-122 under the stimulation of extracellular drugs in living cells and animals. Furthermore, to prove the modularity of the system, we engineered this miCop to tune the expression of the DTA (diphtheria toxin A) gene and showed its powerful capacity to kill cancer cells by inducing apoptosis and cell cycle arrest based on miRNA response. This study provides an effective means to couple miRNA sensing with miRNA-responsive gene modulation, which may open up new diagnostic or therapeutic applications.

Expanding DdCBE-mediated targeting scope to aC motif preference in rat.

An animal model harboring pathogenic mitochondrial DNA (mtDNA) mutations is important to understand the biological links between mtDNA variation and mitochondrial diseases. DdCBE, a DddA-derived cytosine base editor, has been utilized in zebrafish, mice, and rats for tC sequence-context targeting and human mitochondrial disease modeling. However, human pathogenic mtDNA mutations other than the tC context cannot be manipulated. Here, we screened the combination of different DdCBE pairs at pathogenic mtDNA mutation sites with nC (n for a, g, or c) context and identified that the left-G1333C (L1333C) + right G1333N (R1333N) pair could mediate C⋅G-to-T⋅A conversion effectively at aC sites in rat C6 cells. The editing efficiency at disease-associated mtDNA mutation sites within aC context was further confirmed to be up to 67.89% in vivo. Also, the installed disease-associated mtDNA mutations were germline transmittable. Moreover, the edited rats showed impaired cardiac function and mitochondrial function, resembling human mitochondrial disease symptoms. In summary, for the first time, we expanded the DdCBE targeting scope to an aC motif and installed the pathogenic mutation in rats to model human mitochondrial diseases.

A guidebook of spatial transcriptomic technologies, data resources and analysis approaches.

Advances in transcriptomic technologies have deepened our understanding of the cellular gene expression programs of multicellular organisms and provided a theoretical basis for disease diagnosis and therapy. However, both bulk and single-cell RNA sequencing approaches lose the spatial context of cells within the tissue microenvironment, and the development of spatial transcriptomics has made overall bias-free access to both transcriptional information and spatial information possible. Here, we elaborate development of spatial transcriptomic technologies to help researchers select the best-suited technology for their goals and integrate the vast amounts of data to facilitate data accessibility and availability. Then, we marshal various computational approaches to analyze spatial transcriptomic data for various purposes and describe the spatial multimodal omics and its potential for application in tumor tissue. Finally, we provide a detailed discussion and outlook of the spatial transcriptomic technologies, data resources and analysis approaches to guide current and future research on spatial transcriptomics.

Increased mtDNA mutation frequency in oocytes causes epigenetic alterations and embryonic defects.

Mitochondria are essential for female reproductive processes, yet the function of mitochondrial DNA (mtDNA) mutation in oocytes remains elusive. By employing an mtDNA mutator (Polgm) mouse model, we found the fetal growth retardation and placental dysfunction in post-implantation embryos derived from Polgm oocytes. Remarkably, Polgm oocytes displayed the global loss of DNA methylation; following fertilization, zygotic genome experienced insufficient demethylation, along with dysregulation of gene expression. Spindle-chromosome exchange experiment revealed that cytoplasmic factors in Polgm oocytes are responsible for such a deficient epigenetic remodeling. Moreover, metabolomic profiling identified a significant reduction in the α-ketoglutarate (αKG) level in oocytes from Polgm mice. Importantly, αKG supplement restored both DNA methylation state and transcriptional activity in Polgm embryos, consequently preventing the developmental defects. Our findings uncover the important role of oocyte mtDNA mutation in controlling epigenetic reprogramming and gene expression during embryogenesis. αKG deserves further evaluation as a potential drug for treating mitochondrial dysfunction-related fertility decline.

SETD3 Methyltransferase Regulates PLK1 Expression to Promote In Situ Hepatic Carcinogenesis.

Background: The development of a new strategy to overcome chemoresistance to hepatocellular carcinoma (HCC) treatment is a long-standing issue. We have previously found that upregulated SETD3 levels are closely correlated with HCC. This study aims to explore the mechanism underlying how upregulation of SETD3 promotes liver carcinogenesis. Methods: RNA-Sequencing analysis was used to explore the correlation of SETD3 with regulatory targets. In vitro assays including cell proliferation and migration were performed to study the oncogenic roles of SETD3 and PLK1. Western blotting, immunohistochemical staining, and blood biochemical assays were performed to examine protein expression or pathological index in tumor tissues and mice liver tissues. Luciferase reporter system and chromatin immunoprecipitation assays were used to explore the mechanism. Results: We revealed that SETD3 regulates gene expression in subgroups, including cell division, cell proliferation, and cell cycle, in hepatocellular tumor cells. We found that SETD3 upregulation is associated with elevated PLK1 level in both hepatic tumor cells and clinical liver tissues. We further showed that overexpression of SETD3 promoted tumor cell proliferation and migration, whereas inhibition of PLK1 activity attenuated these phenotypes caused by SETD3. By taking advantage of the Sleep Beauty transposase system, we confirmed that upregulated mouse Setd3 promoted hepatic carcinogenesis in situ, but knockdown of mouse Plk1 mitigated Setd3-promoted tumorigenesis in mice. Mechanistically, we showed that SETD3 could be recruited to the promoter of PLK1 gene to facilitate PLK1 transcription. Conclusions: Our data demonstrate that elevated SETD3 may promote HCC by enhancing PLK1 expression, which suggests that SETD3 may act as a potential drug target combined with PLK1 inhibition to treat HCC.

Integrative proteome analysis implicates aberrant RNA splicing in impaired developmental potential of aged mouse oocytes.

Aging has many effects on the female reproductive system, among which decreased oocyte quality and impaired embryo developmental potential are the most important factors affecting female fertility. However, the mechanisms underlying oocyte aging are not yet fully understood. Here, we selected normal reproductively aging female mice and constructed a protein expression profile of metaphase II (MII) oocytes from three age groups. A total of 187 differentially expressed (DE) proteins were identified, and bioinformatics analyses showed that these DE proteins were highly enriched in RNA splicing. Next, RNA-seq was performed on 2-cell embryos from these three age groups, and splicing analysis showed that a large number of splicing events and genes were discovered at this stage. Differentially spliced genes (DSGs) in the two reproductively aging groups versus the younger group were enriched in biological processes related to DNA damage repair/response. Binding motif analysis suggested that PUF60 might be one of the core splicing factors causing a decline in DNA repair capacity in the subsequent development of oocytes from reproductively aging mice, and changing the splicing pattern of its potential downstream DSG Cdk9 could partially mimic phenotypes in the reproductively aging groups. Taken together, our study suggested that the abnormal expression of splicing regulation proteins in aged MII oocytes would affect the splicing of nascent RNA after zygotic genome activation in 2-cell embryos, leading to the production of abnormally spliced transcripts of some key genes associated with DNA damage repair/response, thus affecting the developmental potential of aged oocytes.

Correction: Dynamic visualization of mRNA splicing variants with a transactivating reporter.

Correction for 'Dynamic visualization of mRNA splicing variants with a transactivating reporter' by Si Chen et al., Chem. Commun., 2021, DOI: 10.1039/d1cc02439f.

Dynamic visualization of mRNA splicing variants with a transactivating reporter.

Dynamic changes in intron sequences, with their loss and gain, are poorly detected due to the limited methods for the non-invasive monitoring of the pre-mRNA splicing process. Here, we describe the design of a two-step transcriptional activation (TSTA) reporter for the real-time imaging of the splicing process in living subjects. By taking advantage of the strong transactivating properties of the GAL4-VP16 fusion protein, which can target upstream activation sequence (UAS) elements to boost subsequent firefly luciferase reporter gene expression, we successfully and consistently detected the dynamic pre-mRNA splicing activity in response to exogenous splicing modulators in living cells and animals. Our findings provide a valuable tool for the high-throughput screening of splicing modulators, which could speed up the development of new drugs for the treatment of disordered splicing diseases.

Upregulation of PAIP1 promotes the gallbladder tumorigenesis through regulating PLK1 level.

BACKGROUND: Increasing evidence suggests that elevated expression of polyA-binding protein-interacting protein 1 (PAIP1) is associated with cancer development and progression. However, how PAIP1 promotes gallbladder cancer (GBC) is still unclear. METHODS: Two GBC tissue-derived cell lines, NOZ and GBC-SD cells, were used in this study. Assays of cell proliferation, colony formation, apoptosis, and xenograft tumor model were performed to examine the tumorigenic effects of PAIP1. Immunohistochemical (IHC) staining was used to examine the expression level of PAIP1 in both patient GBC tissues and mouse tumors. Microarray and bioinformatics analysis were used to explore the targets of PAIP1. Quantitative polymerase chain reaction (qPCR) and western blot analysis were used to validate PAIP1-mediated targets. RESULTS: We found that upregulated PAIP1 expression was correlated with GBC. Knockdown of PAIP1 in gallbladder cells alleviated cell proliferation, promoted apoptosis, and inhibited xenograft tumor growth. Gene microarray analysis showed that stable silencing of PAIP1 altered various gene expressions. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis suggested that PAIP1 regulates cell cycle progression. Finally, we found that the PLK1 kinase, a key regulator of cell cycle, was regulated by PAIP1 at the transcriptional and protein levels. PLK1 level was positively correlated with PAIP1 level in both mouse tumors and GBC tissues. PAIP1 interacted with PLK1, and rescue of PAIP1 could recover PLK1 protein level and inhibit apoptosis. CONCLUSIONS: Our data suggest that PAIP1 contributes to GBC progression likely through regulating PLK1 level. Since upregulated PAIP1 expression is positively associated with GBC, PAIP1 may act as a clinical prognostic biomarker of GBC.

Cytosine and adenine deaminase base-editors induce broad and nonspecific changes in gene expression and splicing.

Cytosine or adenine base editors (CBEs or ABEs) hold great promise in therapeutic applications because they enable the precise conversion of targeted base changes without generating of double-strand breaks. However, both CBEs and ABEs induce substantial off-target DNA editing, and extensive off-target RNA single nucleotide variations in transfected cells. Therefore, the potential effects of deaminases induced by DNA base editors are of great importance for their clinical applicability. Here, the transcriptome-wide deaminase effects on gene expression and splicing is examined. Differentially expressed genes (DEGs) and differential alternative splicing (DAS) events, induced by base editors, are identified. Both CBEs and ABEs generated thousands of DEGs and hundreds of DAS events. For engineered CBEs or ABEs, base editor-induced variants had little effect on the elimination of DEGs and DAS events. Interestingly, more DEGs and DAS events are observed as a result of over expressions of cytosine and adenine deaminases. This study reveals a previously overlooked aspect of deaminase effects in transcriptome-wide gene expression and splicing, and underscores the need to fully characterize such effects of deaminase enzymes in base editor platforms.

Single-cell RNA-Seq reveals a highly coordinated transcriptional program in mouse germ cells during primordial follicle formation.

The assembly of primordial follicles in mammals represents one of the most critical processes in ovarian biology. It directly affects the number of oocytes available to a female throughout her reproductive life. Premature depletion of primordial follicles contributes to the ovarian pathology primary ovarian insufficiency (POI). To delineate the developmental trajectory and regulatory mechanisms of oocytes during the process, we performed RNA-seq on single germ cells from newborn (P0.5) ovaries. Three cell clusters were classified which corresponded to three cell states (germ cell cyst, cyst breakdown, and follicle) in the newborn ovary. By Monocle analysis, a uniform trajectory of oocyte development was built with a series of genes showed dynamic changes along the pseudo-timeline. Gene Ontology term enrichment revealed a significant decrease in meiosis-related genes and a dramatic increase in oocyte-specific genes which marked the transition from a germ cell to a functional oocyte. We then established a network of regulons by using single-cell regulatory network inference and clustering (SCENIC) algorithm and identified possible candidate transcription factors that may maintain transcription programs during follicle formation. Following functional studies further revealed the differential regulation of the identified regulon Id2 and its family member Id1, on the establishment of primordial follicle pool by using siRNA knockdown and genetic modified mouse models. In summary, our study systematically reconstructed molecular cascades in oocytes and identified a series of genes and molecular pathways in follicle formation and development.

O-GlcNAcylation of TDP-43 suppresses proteinopathies and promotes TDP-43's mRNA splicing activity.

Pathological TDP-43 aggregation is characteristic of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP); however, how TDP-43 aggregation and function are regulated remain poorly understood. Here, we show that O-GlcNAc transferase OGT-mediated O-GlcNAcylation of TDP-43 suppresses ALS-associated proteinopathies and promotes TDP-43's splicing function. Biochemical and cell-based assays indicate that OGT's catalytic activity suppresses TDP-43 aggregation and hyperphosphorylation, whereas abolishment of TDP-43 O-GlcNAcylation impairs its RNA splicing activity. We further show that TDP-43 mutations in the O-GlcNAcylation sites improve locomotion defects of larvae and adult flies and extend adult life spans, following TDP-43 overexpression in Drosophila motor neurons. We finally demonstrate that O-GlcNAcylation of TDP-43 promotes proper splicing of many mRNAs, including STMN2, which is required for normal axonal outgrowth and regeneration. Our findings suggest that O-GlcNAcylation might be a target for the treatment of TDP-43-linked pathogenesis.