Regulatory Expansion in Mammals of Multivalent hnRNP Assemblies that Globally Control Alternative Splicing.

Alternative splicing (AS) patterns have diverged rapidly during vertebrate evolution, yet the functions of most species- and lineage-specific splicing events are not known. We observe that mammalian-specific AS events are enriched in transcript sequences encoding intrinsically disordered regions (IDRs) of proteins, in particular those containing glycine/tyrosine repeats that mediate formation of higher-order protein assemblies implicated in gene regulation and human disease. These evolutionary changes impact nearly all members of the hnRNP A and D families of RNA binding proteins. Regulation of these events requires formation of unusual, long-range mammalian-specific RNA duplexes. Differential inclusion of the alternative exons controls the formation of tyrosine-dependent multivalent hnRNP assemblies that, in turn, function to globally regulate splicing. Together, our results demonstrate that AS control of IDR-mediated interactions between hnRNPs represents an important and recurring mechanism underlying splicing regulation. Furthermore, this mechanism has expanded the regulatory capacity of mammalian cells.

Systematic analysis of alternative exon-dependent interactome remodeling reveals multitasking functions of gene regulatory factors.

Alternative splicing significantly expands biological complexity, particularly in the vertebrate nervous system. Increasing evidence indicates that developmental and tissue-dependent alternative exons often control protein-protein interactions; yet, only a minor fraction of these events have been characterized. Using affinity purification-mass spectrometry (AP-MS), we show that approximately 60% of analyzed neural-differential exons in proteins previously implicated in transcriptional regulation result in the gain or loss of interaction partners, which in some cases form unexpected links with coupled processes. Notably, a neural exon in Chtop regulates its interaction with the Prmt1 methyltransferase and DExD-Box helicases Ddx39b/a, affecting its methylation and activity in promoting RNA export. Additionally, a neural exon in Sap30bp affects interactions with RNA processing factors, modulating a critical function of Sap30bp in promoting the splicing of <100 nt "mini-introns" that control nuclear RNA levels. AP-MS is thus a powerful approach for elucidating the multifaceted functions of proteins imparted by context-dependent alternative exons.

Genome-scale mapping of DNA damage suppressors through phenotypic CRISPR-Cas9 screens.

To maintain genome integrity, cells must accurately duplicate their genome and repair DNA lesions when they occur. To uncover genes that suppress DNA damage in human cells, we undertook flow-cytometry-based CRISPR-Cas9 screens that monitored DNA damage. We identified 160 genes whose mutation caused spontaneous DNA damage, a list enriched in essential genes, highlighting the importance of genomic integrity for cellular fitness. We also identified 227 genes whose mutation caused DNA damage in replication-perturbed cells. Among the genes characterized, we discovered that deoxyribose-phosphate aldolase DERA suppresses DNA damage caused by cytarabine (Ara-C) and that GNB1L, a gene implicated in 22q11.2 syndrome, promotes biogenesis of ATR and related phosphatidylinositol 3-kinase-related kinases (PIKKs). These results implicate defective PIKK biogenesis as a cause of some phenotypes associated with 22q11.2 syndrome. The phenotypic mapping of genes that suppress DNA damage therefore provides a rich resource to probe the cellular pathways that influence genome maintenance.

A central chaperone-like role for 14-3-3 proteins in human cells.

14-3-3 proteins are highly conserved regulatory proteins that interact with hundreds of structurally diverse clients and act as central hubs of signaling networks. However, how 14-3-3 paralogs differ in specificity and how they regulate client protein function are not known for most clients. Here, we map the interactomes of all human 14-3-3 paralogs and systematically characterize the effect of disrupting these interactions on client localization. The loss of 14-3-3 binding leads to the coalescence of a large fraction of clients into discrete foci in a client-specific manner, suggesting a central chaperone-like function for 14-3-3 proteins. Congruently, the engraftment of 14-3-3 binding motifs to nonclients can suppress their aggregation or phase separation. Finally, we show that 14-3-3s negatively regulate the localization of the RNA-binding protein SAMD4A to cytoplasmic granules and inhibit its activity as a translational repressor. Our work suggests that 14-3-3s have a more prominent role as chaperone-like molecules than previously thought.

A Dynamic Protein Interaction Landscape of the Human Centrosome-Cilium Interface.

The centrosome is the primary microtubule organizing center of the cells and templates the formation of cilia, thereby operating at a nexus of critical cellular functions. Here, we use proximity-dependent biotinylation (BioID) to map the centrosome-cilium interface; with 58 bait proteins we generate a protein topology network comprising >7,000 interactions. Analysis of interaction profiles coupled with high resolution phenotypic profiling implicates a number of protein modules in centriole duplication, ciliogenesis, and centriolar satellite biogenesis and highlights extensive interplay between these processes. By monitoring dynamic changes in the centrosome-cilium protein interaction landscape during ciliogenesis, we also identify satellite proteins that support cilia formation. Systematic profiling of proximity interactions combined with functional analysis thus provides a rich resource for better understanding human centrosome and cilia biology. Similar strategies may be applied to other complex biological structures or pathways.

Identification and functional characterization of transcriptional activators in human cells.

Transcription is orchestrated by thousands of transcription factors (TFs) and chromatin-associated proteins, but how these are causally connected to transcriptional activation is poorly understood. Here, we conduct an unbiased proteome-scale screen to systematically uncover human proteins that activate transcription in a natural chromatin context. By combining interaction proteomics and chemical inhibitors, we delineate the preference of these transcriptional activators for specific co-activators, highlighting how even closely related TFs can function via distinct cofactors. We also identify potent transactivation domains among the hits and use AlphaFold2 to predict and experimentally validate interaction interfaces of two activation domains with BRD4. Finally, we show that many novel activators are partners in fusion events in tumors and functionally characterize a myofibroma-associated fusion between SRF and C3orf62, a potent p300-dependent activator. Our work provides a functional catalog of potent transactivators in the human proteome and a platform for discovering transcriptional regulators at genome scale.

Systematic mapping of nuclear domain-associated transcripts reveals speckles and lamina as hubs of functionally distinct retained introns.

The nucleus is highly compartmentalized through the formation of distinct classes of membraneless domains. However, the composition and function of many of these structures are not well understood. Using APEX2-mediated proximity labeling and RNA sequencing, we surveyed human transcripts associated with nuclear speckles, several additional domains, and the lamina. Remarkably, speckles and lamina are associated with distinct classes of retained introns enriched in genes that function in RNA processing, translation, and the cell cycle, among other processes. In contrast to the lamina-proximal introns, retained introns associated with speckles are relatively short, GC-rich, and enriched for functional sites of RNA-binding proteins that are concentrated in these domains. They are also highly differentially regulated across diverse cellular contexts, including the cell cycle. Thus, our study provides a resource of nuclear domain-associated transcripts and further reveals speckles and lamina as hubs of distinct populations of retained introns linked to gene regulation and cell cycle progression.

A quantitative chaperone interaction network reveals the architecture of cellular protein homeostasis pathways.

Chaperones are abundant cellular proteins that promote the folding and function of their substrate proteins (clients). In vivo, chaperones also associate with a large and diverse set of cofactors (cochaperones) that regulate their specificity and function. However, how these cochaperones regulate protein folding and whether they have chaperone-independent biological functions is largely unknown. We combined mass spectrometry and quantitative high-throughput LUMIER assays to systematically characterize the chaperone-cochaperone-client interaction network in human cells. We uncover hundreds of chaperone clients, delineate their participation in specific cochaperone complexes, and establish a surprisingly distinct network of protein-protein interactions for cochaperones. As a salient example of the power of such analysis, we establish that NUDC family cochaperones specifically associate with structurally related but evolutionarily distinct ß-propeller folds. We provide a framework for deciphering the proteostasis network and its regulation in development and disease and expand the use of chaperones as sensors for drug-target engagement.

Comprehensive interactome profiling of the human Hsp70 network highlights functional differentiation of J domains.

Hsp70s comprise a deeply conserved chaperone family that has a central role in maintaining protein homeostasis. In humans, Hsp70 client specificity is provided by 49 different co-factors known as J domain proteins (JDPs). However, the cellular function and client specificity of JDPs have largely remained elusive. We have combined affinity purification-mass spectrometry (AP-MS) and proximity-dependent biotinylation (BioID) to characterize the interactome of all human JDPs and Hsp70s. The resulting network suggests specific functions for many uncharacterized JDPs, and we establish a role of conserved JDPs DNAJC9 and DNAJC27 in histone chaperoning and ciliogenesis, respectively. Unexpectedly, we find that the J domain of DNAJC27 but not of other JDPs can fully replace the function of endogenous DNAJC27, suggesting a previously unappreciated role for J domains themselves in JDP specificity. More broadly, our work expands the role of the Hsp70-regulated proteostasis network and provides a platform for further discovery of JDP-dependent functions.

A Rad50-null mutation in mouse germ cells causes reduced DSB formation, abnormal DSB end resection and complete loss of germ cells.

The conserved MRE11-RAD50-NBS1/Xrs2 complex is crucial for DNA break metabolism and genome maintenance. Although hypomorphic Rad50 mutation mice showed normal meiosis, both null and hypomorphic rad50 mutation yeast displayed impaired meiosis recombination. However, the in vivo function of Rad50 in mammalian germ cells, particularly its in vivo role in the resection of meiotic double strand break (DSB) ends at the molecular level remains elusive. Here, we have established germ cell-specific Rad50 knockout mouse models to determine the role of Rad50 in mitosis and meiosis of mammalian germ cells. We find that Rad50-deficient spermatocytes exhibit defective meiotic recombination and abnormal synapsis. Mechanistically, using END-seq, we demonstrate reduced DSB formation and abnormal DSB end resection occurs in mutant spermatocytes. We further identify that deletion of Rad50 in gonocytes leads to complete loss of spermatogonial stem cells due to genotoxic stress. Taken together, our results reveal the essential role of Rad50 in mammalian germ cell meiosis and mitosis, and provide in vivo views of RAD50 function in meiotic DSB formation and end resection at the molecular level.

Interactome Rewiring Following Pharmacological Targeting of BET Bromodomains.

Targeting bromodomains (BRDs) of the bromo-and-extra-terminal (BET) family offers opportunities for therapeutic intervention in cancer and other diseases. Here, we profile the interactomes of BRD2, BRD3, BRD4, and BRDT following treatment with the pan-BET BRD inhibitor JQ1, revealing broad rewiring of the interaction landscape, with three distinct classes of behavior for the 603 unique interactors identified. A group of proteins associate in a JQ1-sensitive manner with BET BRDs through canonical and new binding modes, while two classes of extra-terminal (ET)-domain binding motifs mediate acetylation-independent interactions. Last, we identify an unexpected increase in several interactions following JQ1 treatment that define negative functions for BRD3 in the regulation of rRNA synthesis and potentially RNAPII-dependent gene expression that result in decreased cell proliferation. Together, our data highlight the contributions of BET protein modules to their interactomes allowing for a better understanding of pharmacological rewiring in response to JQ1.

DIA-based phosphoproteomics identifies early phosphorylation events in response to EGTA and mannitol in Arabidopsis.

Osmotic stress significantly hampers plant growth and crop yields, emphasizing the need for a thorough comprehension of the underlying molecular responses. Previous research has demonstrated that osmotic stress rapidly induces calcium influx and signaling, along with the activation of a specific subset of protein kinases, notably the Raf-SnRK2 kinase cascades within minutes. However, the intricate interplay between calcium signaling and the activation of RAF-SnRK2 kinase cascades remains elusive. Here in this study, we discovered that Raf-like protein (RAF) kinases undergo hyperphosphorylation in response to osmotic shocks. Intriguingly, treatment with the calcium chelator EGTA robustly activates RAF-SnRK2 cascades, mirroring the effects of osmotic treatment. Utilizing high-throughput DIA-based phosphoproteomics, we unveiled the global impact of EGTA on protein phosphorylation. Beyond the activation of RAFs and sucrose non-fermenting-1-related protein kinase 2s (SnRK2s), EGTA treatment also activates mitogen-activated protein kinase (MAPKs) cascades, Calcium-dependent protein kinases (CDPKs), and receptor-like protein kinases, etc. Through overlapping assays, we identified potential roles of mitogen-activated protein kinase kinase kinase kinases (MAP4Ks) and receptor-like protein kinases in the osmotic-stress-induced activation of RAF-SnRK2 cascades. Our findings illuminate the regulation of phosphorylation and cellular events by Ca2+ signaling, offering insights into the (exocellular) Ca2+ deprivation during early hyperosmolality sensing and signaling.

Rectifying METTL4-Mediated N6-Methyladenine Excess in Mitochondrial DNA Alleviates Heart Failure.

BACKGROUND: Myocardial mitochondrial dysfunction underpins the pathogenesis of heart failure (HF), yet therapeutic options to restore myocardial mitochondrial function are scarce. Epigenetic modifications of mitochondrial DNA (mtDNA), such as methylation, play a pivotal role in modulating mitochondrial homeostasis. However, their involvement in HF remains unclear. METHODS: Experimental HF models were established through continuous angiotensin II and phenylephrine (AngII/PE) infusion or prolonged myocardial ischemia/reperfusion injury. The landscape of N6-methyladenine (6mA) methylation within failing cardiomyocyte mtDNA was characterized using high-resolution mass spectrometry and methylated DNA immunoprecipitation sequencing. A tamoxifen-inducible cardiomyocyte-specific Mettl4 knockout mouse model and adeno-associated virus vectors designed for cardiomyocyte-targeted manipulation of METTL4 (methyltransferase-like protein 4) expression were used to ascertain the role of mtDNA 6mA and its methyltransferase METTL4 in HF. RESULTS: METTL4 was predominantly localized within adult cardiomyocyte mitochondria. 6mA modifications were significantly more abundant in mtDNA than in nuclear DNA. Postnatal cardiomyocyte maturation presented with a reduction in 6mA levels within mtDNA, coinciding with a decrease in METTL4 expression. However, an increase in both mtDNA 6mA level and METTL4 expression was observed in failing adult cardiomyocytes, suggesting a shift toward a neonatal-like state. METTL4 preferentially targeted mtDNA promoter regions, which resulted in interference with transcription initiation complex assembly, mtDNA transcriptional stalling, and ultimately mitochondrial dysfunction. Amplifying cardiomyocyte mtDNA 6mA through METTL4 overexpression led to spontaneous mitochondrial dysfunction and HF phenotypes. The transcription factor p53 was identified as a direct regulator of METTL4 transcription in response to HF-provoking stress, thereby revealing a stress-responsive mechanism that controls METTL4 expression and mtDNA 6mA. Cardiomyocyte-specific deletion of the Mettl4 gene eliminated mtDNA 6mA excess, preserved mitochondrial function, and mitigated the development of HF upon continuous infusion of AngII/PE. In addition, specific silencing of METTL4 in cardiomyocytes restored mitochondrial function and offered therapeutic relief in mice with preexisting HF, irrespective of whether the condition was induced by AngII/PE infusion or myocardial ischemia/reperfusion injury. CONCLUSIONS: Our findings identify a pivotal role of cardiomyocyte mtDNA 6mA and the corresponding methyltransferase, METTL4, in the pathogenesis of mitochondrial dysfunction and HF. Targeted suppression of METTL4 to rectify mtDNA 6mA excess emerges as a promising strategy for developing mitochondria-focused HF interventions.

Oncolytic strategy using new bifunctional HDACs/BRD4 inhibitors against virus-associated lymphomas.

Primary effusion lymphoma (PEL) caused by Kaposi sarcoma-associated herpesvirus (KSHV) is an aggressive malignancy with poor prognosis even under chemotherapy. Currently, there is no specific treatment for PEL therefore requiring new therapies. Both histone deacetylases (HDACs) and bromodomain-containing protein 4 (BRD4) have been found as therapeutic targets for PEL through inducing viral lytic reactivation. However, the strategy of dual targeting with one agent and potential synergistic effects have never been explored. In the current study, we first demonstrated the synergistic effect of concurrently targeting HDACs and BRD4 on KSHV reactivation by using SAHA or entinostat (HDACs inhibitors) and (+)-JQ1 (BRD4 inhibitor), which indicated dual blockage of HDACs/BRD4 is a viable therapeutic approach. We were then able to rationally design and synthesize a series of new small-molecule inhibitors targeting HDACs and BRD4 with a balanced activity profile by generating a hybrid of the key binding motifs between (+)-JQ1 and entinostat or SAHA. Upon two iterative screenings of optimized compounds, a pair of epimers, 009P1 and 009P2, were identified to better inhibit the growth of KSHV positive lymphomas compared to (+)-JQ1 or SAHA alone at low nanomolar concentrations, but not KSHV negative control cells or normal cells. Mechanistic studies of 009P1 and 009P2 demonstrated significantly enhanced viral reactivation, cell cycle arrest and apoptosis in KSHV+ lymphomas through dually targeting HDACs and BRD4 signaling activities. Importantly, in vivo preclinical studies showed that 009P1 and 009P2 dramatically suppressed KSHV+ lymphoma progression with oral bioavailability and minimal visible toxicity. These data together provide a novel strategy for the development of agents for inducing lytic activation-based therapies against these viruses-associated malignancies.

Genome-wide CRISPR screens identify novel regulators of wild-type and mutant p53 stability.

Tumor suppressor p53 (TP53) is frequently mutated in cancer, often resulting not only in loss of its tumor-suppressive function but also acquisition of dominant-negative and even oncogenic gain-of-function traits. While wild-type p53 levels are tightly regulated, mutants are typically stabilized in tumors, which is crucial for their oncogenic properties. Here, we systematically profiled the factors that regulate protein stability of wild-type and mutant p53 using marker-based genome-wide CRISPR screens. Most regulators of wild-type p53 also regulate p53 mutants, except for p53 R337H regulators, which are largely private to this mutant. Mechanistically, FBXO42 emerged as a positive regulator for a subset of p53 mutants, working with CCDC6 to control USP28-mediated mutant p53 stabilization. Additionally, C16orf72/HAPSTR1 negatively regulates both wild-type p53 and all tested mutants. C16orf72/HAPSTR1 is commonly amplified in breast cancer, and its overexpression reduces p53 levels in mouse mammary epithelium leading to accelerated breast cancer. This study offers a network perspective on p53 stability regulation, potentially guiding strategies to reinforce wild-type p53 or target mutant p53 in cancer.

Dynamic transcriptome landscape of developing maize ear.

Seed number and harvesting ability in maize (Zea mays L.) are primarily determined by the architecture of female inflorescence, namely the ear. Therefore, ear morphogenesis contributes to grain yield and as such is one of the key target traits during maize breeding. However, the molecular networks of this highly dynamic and complex grain-bearing inflorescence remain largely unclear. As a first step toward characterizing these networks, we performed a high-spatio-temporal-resolution investigation of transcriptomes using 130 ear samples collected from developing ears with length from 0.1 mm to 19.0 cm. Comparisons of these mRNA populations indicated that these spatio-temporal transcriptomes were clearly separated into four distinct stages stages I, II, III, and IV. A total of 23 793 genes including 1513 transcription factors (TFs) were identified in the investigated developing ears. During the stage I of ear morphogenesis, 425 genes were predicted to be involved in a co-expression network established by eight hub TFs. Moreover, 9714 ear-specific genes were identified in the seven kinds of meristems. Additionally, 527 genes including 59 TFs were identified as especially expressed in ear and displayed high temporal specificity. These results provide a high-resolution atlas of gene activity during ear development and help to unravel the regulatory modules associated with the differentiation of the ear in maize.

Nab-paclitaxel plus S-1 versus nab-paclitaxel plus gemcitabine in patients with advanced pancreatic cancer: a multicenter, randomized, phase II study.

BACKGROUND: Encouraging antitumor activity of nab-paclitaxel plus S-1 (AS) has been shown in several small-scale studies. This study compared the efficacy and safety of AS versus standard-of-care nab-paclitaxel plus gemcitabine (AG) as a first-line treatment for advanced pancreatic cancer (PC). METHODS: In this multicenter, randomized, phase II trial, eligible patients with unresectable, locally advanced, or metastatic PC were recruited and randomly assigned (1:1) to receive AS (nab-paclitaxel 125 mg/m2 on days 1 and 8; S-1 twice daily on days 1 through 14) or AG (nab-paclitaxel 125 mg/m2 on days 1 and 8; gemcitabine 1000 mg/m2 on days 1 and 8) for 6 cycles. The primary endpoint was progression-free survival (PFS). RESULTS: Between July 16, 2019, and September 9, 2022, 62 patients (AS, n = 32; AG, n = 30) were treated and evaluated. With a median follow-up of 8.36 months at preplanned interim analysis (data cutoff, March 24, 2023), the median PFS (8.48 vs 4.47 months; hazard ratio [HR], 0.402; P = .002) and overall survival (OS; 13.73 vs 9.59 months; HR, 0.226; P < .001) in the AS group were significantly longer compared to the AG group. More patients had objective response in the AS group than AG group (37.50% vs 6.67%; P = .005). The most common grade 3-4 adverse events were neutropenia and leucopenia in both groups, and gamma glutamyl transferase increase was observed only in the AG group. CONCLUSION: The first-line AS regimen significantly extended both PFS and OS of Chinese patients with advanced PC when compared with the AG regimen, with a comparable safety profile. (ClinicalTrials.gov Identifier: NCT03636308).

Enhanced Aggregation-Induced Delayed Electrochemiluminescence Triggered by Spatial Perturbation of Organic Dots.

Development of highly efficient, heavy-metal-free electrochemiluminescence (ECL) materials is attractive but still challenging. Herein, we report an aggregation-induced delayed ECL (AIDECL) active organic dot (OD) composed of a tert-butoxy-group-substituted benzophenone-dimethylacridine compound, which shows high ECL efficiency. The resultant ODs exhibit 2.1-fold higher ECL efficiency compared to control AIDECL-active ODs. Molecular stacking combined with theoretical calculations suggests that tert-butoxy groups effectively participate in the intermolecular interactions, further inhibiting the molecular motions in the aggregated states and thus accelerating radiative decay. On the basis of these ODs exhibiting excellent ECL performance, a proof-of-concept biosensor is constructed for the detection of miR-16 associated with Alzheimer's disease, which demonstrates excellent detection ability with the limit of detection of 1.7 fM. This work provides a new approach to improve the ECL efficiency and enriches the fundamental understanding of the structure-property relationship.

Regulating Grafting Density to Realize High-Areal-Capacity Silicon Submicroparticle Anodes Under Ultralow Binder Content.

Grafted biopolymer binders are demonstrated to improve the processability and cycling stability of the silicon (Si) nanoparticle anodes. However, there is little systematical exploration regarding the relationship between grafting density and performance of grafted binder for Si anodes, especially when Si particles exceed the critical breaking size. Herein, a series of guar gum grafted polyacrylamide (GP) binders with different grafting densities are designed and prepared to determine the optimal grafting density for maximizing the electrochemical performance of Si submicroparticle (SiSMP) anodes. Among various GP binders, GP5 with recommended grafting density demonstrates the strongest adhesion strength, best mechanical properties, and highest intrinsic ionic conductivity. These characteristics enable the SiSMP electrodes to sustain the electrode integrity and accelerate lithium-ion transport kinetics during cycling, resulting in high capacity and stable cyclability. The superior role of GP5 binder in enabling robust structure and stable interface of SiSMP electrodes is revealed through the PeakForce atomic force microscopy and in situ differential electrochemical mass spectrometry. Furthermore, the stable cyclabilities of high-loading SiSMP@GP5 electrode with ultralow GP5 content (1 wt%) at high areal capacity as well as the good cyclability of Ah-level LiNi0.8Co0.1Mn0.1O2/SiSMP@GP5 pouch cell strongly confirms the practical viability of the GP5 binder.

Human endogenous retrovirus type K encoded Np9 oncoprotein induces DNA damage response.

Human endogenous retrovirus sequences (HERVs) constitute up to 8% of the human genome, yet not all HERVs remain silent passengers within our genomes. Some HERVs, especially the HERV type K (HERV-K), have been found to be frequently transactivated in a variety of inflammatory diseases and human cancers. Np9, a 9-kDa HERV-K encoded protein, has been reported as an oncoprotein and found present in a variety of tumors and transformed cells. In the current study, we for the first time reported that ectopic expression of Np9 protein was able to induce DNA damage response from host cells especially through upregulation of γH2AX. Furthermore, we found that direct knockdown of Np9 by RNAi in Kaposi's Sarcoma-associated herpesvirus (KSHV) infected cells effectively reduced LANA expression, the viral major latent oncoprotein in vitro and in vivo, which may represent a novel strategy against virus-associated malignancies.