The Phlebovirus Ribonucleoprotein: An Overview.

In negative strand RNA viruses, ribonucleoproteins, not naked RNA, constitute the template used by the large protein endowed with polymerase activity for replicating and transcribing the viral genome. Here we give an overview of the structures and functions of the ribonucleoprotein from phleboviruses. The nucleocapsid monomer, which constitutes the basic structural unit, possesses a flexible arm allowing for a conformational switch between a closed monomeric state and the formation of a polymeric filamentous structure competent for viral RNA binding and encapsidation in the open state of N. The modes of N-N oligomerization as well as interactions with vRNA are described. Finally, recent advances in tomography open exciting perspectives for a more complete understanding of N-L interactions and the design of specific antiviral compounds.

Trim21-mediated CCT2 ubiquitination suppresses malignant progression and promotes CD4+T cell activation in breast cancer.

Breast cancer remains a significant global health challenge, and its mechanisms of progression and metastasis are still not fully understood. In this study, analysis of TCGA and GEO datasets revealed a significant increase in CCT2 expression in breast cancer tissues, which was associated with poor prognosis in breast cancer patients. Functional analysis revealed that CCT2 promoted breast cancer growth and metastasis through activation of the JAK2/STAT3 signaling pathway. Additionally, the E3 ubiquitin ligase Trim21 facilitated CCT2 ubiquitination and degradation, significantly reversing the protumor effects of CCT2. Most interestingly, we discovered that exosomal CCT2 derived from breast cancer cells suppressed the activation and proinflammatory cytokine secretion of CD4+ T cell. Mechanistically, exosomal CCT2 constrained Ca2+-NFAT1 signaling, thereby reducing CD40L expression on CD4+ T cell. These findings highlight CCT2 upregulation as a potential driver of breast cancer progression and immune evasion. Our study provides new insights into the molecular mechanisms underlying breast cancer progression, suggesting that CCT2 is a promising therapeutic target and prognostic predictor for breast cancer.

FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion.

The ubiquitin-like modifier FAT10 is upregulated under pro-inflammatory conditions, targets its substrates for proteasomal degradation and functions as a negative regulator of the type-I IFN response. Influenza A virus infection upregulates the production of type-I IFN and the expression of the E3 ligase TRIM21, which regulates type-I IFN production in a positive feedback manner. In this study, we show that FAT10 becomes covalently conjugated to TRIM21 and that this targets TRIM21 for proteasomal degradation. We further show that the coiled-coil and PRYSPRY domains of TRIM21 and the C-terminal diglycine motif of FAT10 are important for the TRIM21-FAT10 interaction. Moreover, upon influenza A virus infection and in the presence of FAT10 the total ubiquitination of TRIM21 is reduced and our data reveal that the FAT10-mediated degradation of TRIM21 diminishes IFNß production. Overall, this study provides strong evidence that FAT10 down-regulates the antiviral type-I IFN production by modulating additional molecules of the RIG-I signaling pathway besides the already published OTUB1. In addition, we elucidate a novel mechanism of FAT10-mediated proteasomal degradation of TRIM21 that regulates its stability.

Pseudorabies virus tegument protein US2 antagonizes antiviral innate immunity by targeting cGAS-STING signaling pathway.

Background: The cGAS-STING axis-mediated type I interferon pathway is a crucial strategy for host defense against DNA virus infection. Numerous evasion strategies developed by the pseudorabies virus (PRV) counteract host antiviral immunity. To what extent PRV-encoded proteins evade the cGAS-STING signaling pathway is unknown. Methods: Using US2 stably expressing cell lines and US2-deficient PRV model, we revealed that the PRV tegument protein US2 reduces STING protein stability and downregulates STING-mediated antiviral signaling. Results: To promote K48-linked ubiquitination and STING degradation, US2 interacts with the LBD structural domain of STING and recruits the E3 ligase TRIM21. TRIM21 deficiency consistently strengthens the host antiviral immune response brought on by PRV infection. Additionally, US2-deficient PRV is less harmful in mice. Conclusions: Our study implies that PRV US2 inhibits IFN signaling by a new mechanism that selectively targets STING while successfully evading the host antiviral response. As a result, the present study reveals a novel strategy by which PRV evades host defense and offers explanations for why the Bartha-K61 classical vaccine strain failed to offer effective defense against PRV variant strains in China, indicating that US2 may be a key target for developing gene-deficient PRV vaccines.

TNKS1BP1 facilitates ubiquitination of CNOT4 by TRIM21 to promote hepatocellular carcinoma progression and immune evasion.

Immune checkpoint inhibitors, particularly PD-1/PD-L1 blockades, have been approved for unresectable hepatocellular carcinoma (HCC). However, high resistance rates still limit their efficacy, highlighting the urgent need to understand the underlying mechanisms and develop strategies for overcoming the resistance. In this study, tankyrasel binding protein 1 (TNKS1BP1) was found to interact with tripartite motif containing 21 (TRIM21) and mediated the ubiquitination of CCR4-NOT transcription complex subunit 4 (CNOT4) at the K239 residue via K48 and K6 linkage, which was essential for its tumorigenesis function. Autophagy and lipid reprogramming were identified as two possible mechanisms underlying the pro-tumor effect of TNKS1BP1. Upregulated TNKS1BP1 inhibited autophagy while induced lipid accumulation by inhibiting the JAK2/STAT3 pathway upon the degradation of CNOT4 in HCC. Importantly, knocking down TNKS1BP1 synergized with anti-PD-L1 treatment by upregulating PD-L1 expression on tumor cells via the JAK2/STAT3 pathway, and remodeling the tumor microenvironment by increasing infiltration of tumor-infiltrating lymphocytes as well as augmenting the effect of cytotoxic T lymphocytes. In conclusion, this study identified TNKS1BP1 as a predictive biomarker for patient prognosis and a promising therapeutic target to overcome anti-PD-L1 resistance in HCC.

LARP3, LARP7, and MePCE are involved in the early stage of human telomerase RNA biogenesis.

Human telomerase assembly is a highly dynamic process. Using biochemical approaches, we find that LARP3 and LARP7/MePCE are involved in the early stage of human telomerase RNA (hTR) and that their binding to RNA is destabilized when the mature form is produced. LARP3 plays a negative role in preventing the processing of the 3'-extended long (exL) form and the binding of LARP7 and MePCE. Interestingly, the tertiary structure of the exL form prevents LARP3 binding and facilitates hTR biogenesis. Furthermore, low levels of LARP3 promote hTR maturation, increase telomerase activity, and elongate telomeres. LARP7 and MePCE depletion inhibits the conversion of the 3'-extended short (exS) form into mature hTR and the cytoplasmic accumulation of hTR, resulting in telomere shortening. Taken together our data suggest that LARP3 and LARP7/MePCE mediate the processing of hTR precursors and regulate the production of functional telomerase.

Enhanced binding of guanylated poly(A) RNA by the LaM domain of LARP1.

La-related proteins (LARPs) are a family of RNA-binding proteins that share a conserved La motif (LaM) domain. LARP1 plays a role in regulating ribosomal protein synthesis and stabilizing mRNAs and has a unique structure without an RNA binding RRM domain adjoining the LaM domain. In this study, we investigated the physical basis for LARP1 specificity for poly(A) sequences and observed an unexpected bias for sequences with single guanines. Multiple guanine substitutions did not increase the affinity, demonstrating preferential recognition of singly guanylated sequences. We also observed that the cyclic di-nucleotides in the cCAS/STING pathway, cyclic-di-GMP and 3',3'-cGAMP, bound with sub-micromolar affinity. Isothermal titration measurements were complemented by high-resolution crystal structures of the LARP1 LaM with six different RNA ligands, including two stereoisomers of a phosphorothioate linkage. The selectivity for singly substituted poly(A) sequences suggests LARP1 may play a role in the stabilizing effect of poly(A) tail guanylation. [Figure: see text].

LARP7 Contributes to Glucose-Induced Cardiac Dysfunction, Apoptosis and Fibrosis by Inhibiting the Degradation of STING.

BACKGROUND: Diabetic cardiomyopathy (DCM) is an important cause of heart failure in diabetic patients. The aim of this study was to investigate the pathogenesis of DCM and to identify potential therapeutic targets. METHODS: A mouse model of type 1 DCM was constructed by continuous intraperitoneal injection of streptozotocin (STZ). Systolic and diastolic functions were measured by ultrasound. The expression of La-related protein 7 (LARP7), the stimulator of interferon genes (STING) pathway and light chain 3 (LC3) in myocardial tissue was detected by Western blot and immunofluorescence analyses. Neonatal mouse ventricular cardiomyocytes (NMVCMs) were isolated and cultured. An in vitro type 1 diabetes mellitus (T1DM) model was established by treatment with high glucose. Knockdown/overexpression of LARP7 and STING was achieved by adenovirus transduction, C-176 (a potent covalent inhibitor of STING), and plasmid transfection. The expression, activation, and localization of STING and LARP7 in cardiomyocytes was evaluated, as well as the interaction between the two. The effect of this interaction on the STING-dependent autophagy‒lysosomal pathway was also explored. In addition, the fibrosis and apoptosis of cardiomyocytes were evaluated. RESULTS: High glucose was found to increase the expression and activation of STING and LARP7 in mouse myocardial tissue. This was accompanied by myocardial fibrosis, impaired autophagy degradation function and impaired cardiac function. These findings were further confirmed by in vitro experiments. High glucose caused LARP7 to translocate from the nucleus to the cytoplasm, where it interacted with accumulated STING to inhibit its degradation. Inhibition of STING or LARP7 expression significantly improved myocardial injury induced by high glucose. CONCLUSIONS: Targeted inhibition of LARP7 or STING expression may be a potential therapeutic strategy for the treatment of DCM.

Highly efficient CRISPR/Cas9-RNP mediated CaPAD1 editing in protoplasts of three pepper (Capsicum annuum L.) cultivars.

Parthenocarpy, characterized by seedless fruit development without pollination or fertilization, offers the advantage of consistent fruit formation, even under challenging conditions such as high temperatures. It can be induced by regulating auxin homeostasis; PAD1 (PARENTAL ADVICE-1) is an inducer of parthenocarpy in Solanaceae plants. However, precise editing of PAD1 is not well studied in peppers. Here, we report a highly efficient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) for CaPAD1 editing in three valuable cultivars of pepper (Capsicum annuum L.): Dempsey, a gene-editable bell pepper; C15, a transformable commercial inbred line; and Younggo 4, a Korean landrace. To achieve the seedless pepper trait under high temperatures caused by unstable climate change, we designed five single guide RNAs (sgRNAs) targeting the CaPAD1 gene. We evaluated the in vitro on-target activity of the RNP complexes in three cultivars. Subsequently, we introduced five CRISPR/Cas9-RNP complexes into protoplasts isolated from three pepper leaves and compared indel frequencies and patterns through targeted deep sequencing analyses. We selected two sgRNAs, sgRNA2 and sgRNA5, which had high in vivo target efficiencies for the CaPAD1 gene across the three cultivars and were validated as potential off-targets in their genomes. These findings are expected to be valuable tools for developing new seedless pepper cultivars through precise molecular breeding of recalcitrant crops in response to climate change.

Exploring non-viral methods for the delivery of CRISPR-Cas ribonucleoprotein to hematopoietic stem cells.

Gene manipulation of hematopoietic stem cells (HSCs) using the CRISPR/Cas system as a potent genome editing tool holds immense promise for addressing hematologic disorders. An essential hurdle in advancing this treatment lies in effectively delivering CRISPR/Cas to HSCs. While various delivery formats exist, Ribonucleoprotein complex (RNP) emerges as a particularly efficient option. RNP complexes offer enhanced gene editing capabilities, devoid of viral vectors, with rapid activity and minimized off-target effects. Nevertheless, novel delivery methods such as microfluidic-based techniques, filtroporation, nanoparticles, and cell-penetrating peptides are continually evolving. This study aims to provide a comprehensive review of these methods and the recent research on delivery approaches of RNP complexes to HSCs.

Ro60-Roles in RNA Processing, Inflammation, and Rheumatic Autoimmune Diseases.

The Ro60/SSA2 autoantigen is an RNA-binding protein and a core component of nucleocytoplasmic ribonucleoprotein (RNP) complexes. Ro60 is essential in RNA metabolism, cell stress response pathways, and cellular homeostasis. It stabilises and mediates the quality control and cellular distribution of small RNAs, including YRNAs (for the 'y' in 'cytoplasmic'), retroelement transcripts, and misfolded RNAs. Ro60 transcriptional dysregulation or loss of function can result in the generation and release of RNA fragments from YRNAs and other small RNAs. Small RNA fragments can instigate an inflammatory cascade through endosomal toll-like receptors (TLRs) and cytoplasmic RNA sensors, which typically sense pathogen-associated molecular patterns, and mount the first line of defence against invading pathogens. However, the recognition of host-originating RNA moieties from Ro60 RNP complexes can activate inflammatory response pathways and compromise self-tolerance. Autoreactive B cells may produce antibodies targeting extracellular Ro60 RNP complexes. Ro60 autoantibodies serve as diagnostic markers for various autoimmune diseases, including Sjögren's disease (SjD) and systemic lupus erythematosus (SLE), and they may also act as predictive markers for anti-drug antibody responses among rheumatic patients. Understanding Ro60's structure, function, and role in self-tolerance can enhance our understanding of the underlying molecular mechanisms of autoimmune conditions.

Polysome collapse and RNA condensation fluidize the cytoplasm.

The cell interior is packed with macromolecules of mesoscale size, and this crowded milieu significantly influences cellular physiology. Cellular stress responses almost universally lead to inhibition of translation, resulting in polysome collapse and release of mRNA. The released mRNA molecules condense with RNA-binding proteins to form ribonucleoprotein (RNP) condensates known as processing bodies and stress granules. Here, we show that polysome collapse and condensation of RNA transiently fluidize the cytoplasm, and coarse-grained molecular dynamic simulations support this as a minimal mechanism for the observed biophysical changes. Increased mesoscale diffusivity correlates with the efficient formation of quality control bodies (Q-bodies), membraneless organelles that compartmentalize misfolded peptides during stress. Synthetic, light-induced RNA condensation also fluidizes the cytoplasm. Together, our study reveals a functional role for stress-induced translation inhibition and formation of RNP condensates in modulating the physical properties of the cytoplasm to enable efficient response of cells to stress conditions.

m6A modification of lncRNA ABHD11-AS1 promotes colorectal cancer progression and inhibits ferroptosis through TRIM21/IGF2BP2/ FOXM1 positive feedback loop.

Long non-coding RNA (lncRNA) is closely related to a variety of human cancers, which may provide huge potential biomarkers for cancer diagnosis and treatment. However, the aberrant expression of most lncRNAs in colorectal cancer (CRC) remains elusive. This study aims to explore the clinical significance and potential mechanism of lncRNA ABHD11 antisense RNA 1 (ABHD11-AS1) in the colorectal cancer. Here, we demonstrated that lncRNA ABHD11-AS1 is high-expressed in colorectal cancer (CRC) patients, and strongly related with poor prognosis. Functionally, ABHD11-AS1 suppresses ferroptosis and promotes proliferation and migration in CRC both in vitro and in vivo. Mechanically, lncRNA ABHD11-AS1 interacted with insulin-like growing factor 2 mRNA-binding protein 2 (IGF2BP2) to enhance FOXM1 stability, forming an ABHD11-AS1/FOXM1 positive feedback loop. E3 ligase tripartite motif containing 21 (TRIM21) promotes the degradation of IGF2BP2 via the K48-ubiquitin-lysosome pathway and ABHD11-AS1 promotes the interaction between IGF2BP2 and TRIM21 as scaffold platform. Furthermore, N6 -adenosine-methyltransferase-like 3 (METTL3) upregulated the stabilization of ABHD11-AS1 through the m6A reader IGF2BP2. Our study highlights ABHD11-AS1 as a significant regulator in CRC and it may become a potential target in future CRC treatment.

TRIM21 and Fc-engineered antibodies: decoding its complex antibody binding mode with implications for viral neutralization.

TRIM21 is a pivotal effector in the immune system, orchestrating antibody-mediated responses and modulating immune signaling. In this comprehensive study, we focus on the interaction of TRIM21 with Fc engineered antibodies and subsequent implications for viral neutralization. Through a series of analytical techniques, including biosensor assays, mass photometry, and electron microscopy, along with structure predictions, we unravel the intricate mechanisms governing the interplay between TRIM21 and antibodies. Our investigations reveal that the TRIM21 capacity to recognize, bind, and facilitate the proteasomal degradation of antibody-coated viruses is critically dependent on the affinity and avidity interplay of its interactions with antibody Fc regions. We suggest a novel binding mechanism, where TRIM21 binding to one Fc site results in the detachment of PRYSPRY from the coiled-coil domain, enhancing mobility due to its flexible linker, thereby facilitating the engagement of the second site, resulting in avidity due to bivalent engagement. These findings shed light on the dual role of TRIM21 in antiviral immunity, both in recognizing and directing viruses for intracellular degradation, and demonstrate its potential for therapeutic exploitation. The study advances our understanding of intracellular immune responses and opens new avenues for the development of antiviral strategies and innovation in tailored effector functions designed to leverage TRIM21s unique binding mode.

The MGF300-2R Protein of African Swine Fever Virus Promotes IKKß Ubiquitination by Recruiting the E3 Ubiquitin Ligase TRIM21.

African swine fever (ASF) is an acute, hemorrhagic, highly contagious disease in pigs caused by African swine fever virus (ASFV). Our previous study identified that the ASFV MGF300-2R protein functions as a virulence factor and found that MGF300-2R degrades IKKß via selective autophagy. However, the E3 ubiquitin ligase responsible for IKKß ubiquitination during autophagic degradation still remains unknown. In order to solve this problem, we first pulled down 328 proteins interacting with MGF300-2R through immunoprecipitation-mass spectrometry. Next, we analyzed and confirmed the interaction between the E3 ubiquitin ligase TRIM21 and MGF300-2R and demonstrated the catalytic role of TRIM21 in IKKß ubiquitination. Finally, we indicated that the degradation of IKKß by MGF300-2R was dependent on TRIM21. In summary, our results indicate TRIM21 is the E3 ubiquitin ligase involved in the degradation of IKKß by MGF300-2R, thereby augmenting our understanding of the functions of MGF300-2R and offering insights into the rational design of live attenuated vaccines and antiviral strategies against ASF.

[TRIM21 Inhibits the Proliferation and Migration of Lung Adenocarcinoma Cells 
by Interacting with ZSWIM1].

BACKGROUND: Lung adenocarcinoma (LUAD) is a highly morbid and fatal cancer. Despite advancements in modern medical treatment, the 5-year survival rate of patients remains suboptimal. Our previous study revealed that zinc finger SWIM-type containing 1 (ZSWIM1), a novel protein, promotes the proliferation, migration, and invasion of LUAD cells. The aim of this study is to investigate the impact of E3 ubiquitin ligase tripartite motif protein 21 (TRIM21) on ZSWIM1-mediated cell proliferation and migration. METHODS: The interaction and co-localization between TRIM21 and ZSWIM1 were verified using co-immunoprecipitation (Co-IP) and immunofluorescence (IF). The effects of TRIM21 and ZSWIM1 on the proliferation and migration of LUAD cells were assessed through MTT and Transwell assays, respectively. Western blot (WB) analysis was conducted to evaluate the impact of TRIM21 and ZSWIM1 on the expression of epithelial-mesenchymal transition (EMT) markers in LUAD cells. The influence of TRIM21 on the ubiquitination of ZSWIM1 was examined using Co-IP combined with WB. RESULTS: TRIM21 was found to interact and co-localize with ZSWIM1. Overexpression of TRIM21 inhibited the proliferation and migration of LUAD cells. Overexpression of TRIM21 reduced the promoting effect of ZSWIM1 on the proliferation, migration, and invasion of lung adenocarcinoma cells, and reversed the impact of ZSWIM1 on the expression of E-cadherin and Vimentin. Conversely, knockdown of TRIM21 further enhanced the promoting effect of ZSWIM1 on the proliferation and migration of LUAD cells. Mechanistically, we observed that overexpression of TRIM21 significantly enhanced the ubiquitination level of ZSWIM1, leading to a decrease in ZSWIM1 protein expression. CONCLUSIONS: TRIM21 binds to and promotes the ubiquitination of ZSWIM1, resulting in reduced protein expression of ZSWIM1, which leads to the inhibition of ZSWIM1-mediated promotion of proliferation, migration, and invasion in LUAD cells.

Deoxynivalenol induces m6A-mediated upregulation of p21 and growth arrest of mouse hippocampal neuron cells in vitro.

Hippocampal neurons maintain the ability of proliferation throughout life to support neurogenesis. Deoxynivalenol (DON) is a mycotoxin that exhibits brain toxicity, yet whether and how DON affects hippocampal neurogenesis remains unknown. Here, we use mouse hippocampal neuron cells (HT-22) as a model to illustrate the effects of DON on neuron proliferation and to explore underlying mechanisms. DON exposure significantly inhibits the proliferation of HT-22 cells, which is associated with an up-regulation of cell cycle inhibitor p21 at both mRNA and protein levels. Global and site-specific m6A methylation levels on the 3'UTR of p21 mRNA are significantly increased in response to DON treatment, whereas inhibition of m6A hypermethylation significantly alleviates DON-induced cell cycle arrest. Further mechanistic studies indicate that the m6A readers YTHDF1 and IGF2BP1 are responsible for m6A-mediated increase in p21 mRNA stability. Meanwhile, 3'UTR of E3 ubiquitin ligase TRIM21 mRNA is also m6A hypermethylated, and another m6A reader YTHDF2 binds to the m6A sites, leading to decreased TRIM21 mRNA stability. Consequently, TRIM21 suppression impairs ubiquitin-mediated p21 protein degradation. Taken together, m6A-mediated upregulation of p21, at both post-transcriptional and post-translational levels, contributes to DON-induced inhibition of hippocampal neuron proliferation. These results may provide new insights for epigenetic therapy of neurodegenerative diseases.

Cationic lipid nanoparticle-mediated delivery of a Cas9/crRNA ribonucleoprotein complex for transgene-free editing of the citrus plant genome.

KEY MESSAGE: A lipofectamine-mediated transfection protocol for DNA-free genome editing of citrus protoplast cells using a Cas9/gRNA ribonucleoprotein (RNP) complex resulted in the production of transgene free genome edited citrus.

RNP granules in ALS and neurodegeneration: From multifunctional membraneless organelles to therapeutic opportunities.

Amyotrophic lateral sclerosis (ALS) and related neurodegenerative diseases are characterised by dysfunction of a host of RNA-binding proteins (RBPs) and a severely disrupted RNA metabolism. Recently, RBP-harbouring phase-separated complexes, ribonucleoprotein (RNP) granules, have come into the limelight as "crucibles" of neuronal pathology in ALS. RNP granules are indispensable for the multitude of regulatory processes underlying cellular RNA metabolism and serve as critical organisers of cellular biochemistry. Neurons, highly specialised cells, heavily rely on RNP granules for efficient trafficking, signalling and stress responses. Multiple RNP granule components, primarily RBPs such as TDP-43 and FUS, are affected by ALS mutations. However, even in the absence of mutations, RBP proteinopathies represent pathophysiological hallmarks of ALS. Given the high local concentrations of RBPs and RNAs, their weakened or enhanced interactions within RNP granules disrupt their homeostasis. Thus, the physiological process of phase separation and RNP granule formation, vital for maintaining the high-functioning state of neuronal cells, becomes their Achilles heel. Here, we will review the recent literature on the causes and consequences of abnormal RNP granule functioning in ALS and related disorders. In particular, we will summarise the evidence for the network-level dysfunction of RNP granules in these conditions and discuss considerations for therapeutic interventions to target RBPs, RNP granules and their network as a whole.

1H, 13C, and 15N resonance assignments of the La Motif of the human La-related protein 1.

Human La-related protein 1 (HsLARP1) is involved in post-transcriptional regulation of certain 5' terminal oligopyrimidine (5'TOP) mRNAs as well as other mRNAs and binds to both the 5'TOP motif and the 3'-poly(A) tail of certain mRNAs. HsLARP1 is heavily involved in cell proliferation, cell cycle defects, and cancer, where HsLARP1 is significantly upregulated in malignant cells and tissues. Like all LARPs, HsLARP1 contains a folded RNA binding domain, the La motif (LaM). Our current understanding of post-transcriptional regulation that emanates from the intricate molecular framework of HsLARP1 is currently limited to small snapshots, obfuscating our understanding of the full picture on HsLARP1 functionality in post-transcriptional events. Here, we present the nearly complete resonance assignment of the LaM of HsLARP1, providing a significant platform for future NMR spectroscopic studies.