Targeting protein condensation in cGAS-STING signaling pathway.

The cGAS-STING signaling pathway plays a pivotal role in sensing cytosolic DNA and initiating innate immune responses against various threats, with disruptions in this pathway being associated with numerous immune-related disorders. Therefore, precise regulation of the cGAS-STING signaling is crucial to ensure appropriate immune responses. Recent research, including ours, underscores the importance of protein condensation in driving the activation and maintenance of innate immune signaling within the cGAS-STING pathway. Consequently, targeting condensation processes in this pathway presents a promising approach for modulating the cGAS-STING signaling and potentially managing associated disorders. In this review, we provide an overview of recent studies elucidating the role and regulatory mechanism of protein condensation in the cGAS-STING signaling pathway while emphasizing its pathological implications. Additionally, we explore the potential of understanding and manipulating condensation dynamics to develop novel strategies for mitigating cGAS-STING-related disorders in the future.

Depression Exacerbates Hepatic Steatosis in C57BL/6J Mice by Activating the Hypothalamic-Pituitary-Adrenal Axis.

BACKGROUND/AIM: Depression is associated with metabolic disorders, including non-alcoholic fatty liver disease (NAFLD). However, the mechanisms underlying the interaction between them are still poorly known. MATERIALS AND METHODS: In this study, mice on a choline deficiency, L-amino acid-defined, high-fat diet (CDAHFD) developing steatosis were challenged with chronic restraint stress (CRS), a protocol widely used to induce depression. The development of depression and steatosis was evaluated using histopathology analysis, ELISA, q-PCR and Western Blot. RESULTS: The contribution of the activated HPA axis to hepatic steatosis progress was fully established, which was validated using a hepatocyte model. Histopathological and biochemical analysis indicated that steatosis was exacerbated by CRS challenge, and behavioral tests indicated that the mice developed depression. Among the screened endocrinal pathways, the hypothalamic-pituitary-adrenal (HPA) axis was significantly activated and the synergistic effect of CDAHFD and CRS in activating the HPA axis was observed. In the hypothalamus, expression of corticotropin-releasing hormone (CRH) was increased by 86% and the protein levels of hypothalamic CRH were upregulated by 25% to 33% by CRS treatment. Plasma CRH levels were elevated by 45-56% and plasma adrenocorticotropic hormone (ACTH) levels were elevated by 29-58% by CRS treatment. In the liver, target genes of the HPA axis were activated, accompanied by disruption of the lipid metabolism and progression of steatohepatitis. The lipid metabolism in the Hepa1-6 cell line treated with endogenous corticosterone (CORT) was in accordance with the aforementioned in vivo responses. CONCLUSION: Depression aggravated hepatic steatosis in CDAHFD-fed mice by activating the HPA axis. The risk of NAFLD development should be fully considered in depressive patients and improvement of psychotic disorders could be an etiological treatment strategy for them.

Construction and validation of a prognostic signature based on anoikis-related lncRNAs in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is the most common type of lung cancer and is characterized by a high death rate and a poor prospect for survival. Anoikis, which is a kind of programmed cell apoptosis, is an important factor in the advancement of tumors. Nonetheless, the function of anoikis-related lncRNAs (ARLRs) in LUAD is still not well understood. The TCGA database was queried for genomic and clinical information. A prognostic signature for ARLRs was established via the use of coexpression analysis and Cox regression. Validation of the model's accuracy was conducted utilizing K-M curves and receiver operating characteristic (ROC) curves, and the signature was utilized to develop a nomogram. LncRNAs were implicated in the progression of tumors, as determined by functional enrichment analysis. There was an improvement in prognosis, increased immune cell infiltration, and higher immune scores among the low-risk patients. Additionally, we found that the two groups had varied anticancer drug sensitivities, which could help guide treatment. The impact of one ARLR, AC026355.2, on migration and invasion was validated by in vitro experiments in LUAD cells. Herein, a new lncRNA signature associated with anoikis was identified and estimated, potentially serving as a prognostic indicator for LUAD patients.

The association between diverse serum folate with MAFLD and liver fibrosis based on NHANES 2017-2020.

Background: Metabolically Associated Fatty Liver Disease (MAFLD) marks a progression from the previous paradigm of Non-Alcoholic Fatty Liver Disease (NAFLD), presenting a redefined diagnostic framework that accentuates metabolic factors while recognizing non-alcoholic contributors. In our investigation, our principal aim was to scrutinize the conceivable correlation between diverse serum folate levels and the prevalence of MAFLD and liver fibrosis. Methods: In our investigation, we conducted an extensive analysis utilizing data derived from the National Health and Nutrition Examination Survey (NHANES) across the years 2017-2020. We aimed to investigate the association between different serum folate concentrations and the prevalence of MAFLD and liver fibrosis by comprehensive multivariate analysis. This analytical approach considered various variables, encompassing sociodemographic characteristics, lifestyle factors, hypertension, and diabetes. By including these potential confounders in our analysis, we aimed to ensure the stability of the findings regarding the association between different serum folate concentrations and the development of MAFLD and liver fibrosis. Results: In our investigation, we utilized multiple linear regression models to thoroughly analyze the data, revealing noteworthy insights. Evidently, elevated levels of both total folate and 5-MTHF exhibited a distinct negative correlation with CAP, while 5-MTHF demonstrated a notable negative correlation with LSM. Furthermore, multiple logistic regression models were employed for an in-depth examination of the data. As the concentrations of total folate and 5-MTHF in the serum increased, a substantial decrease in the likelihood of MAFLD and liver fibrosis occurrence was observed. Conclusion: The findings of this investigation robustly suggest the prevalence of MAFLD and liver fibrosis decreased significantly with the increase of serum concentrations of total folate and 5-MTHF.

Nuclear microRNA-mediated transcriptional control determines adult microglial homeostasis and brain function.

Microglia are versatile regulators in brain development and disorders. Emerging evidence links microRNA (miRNA)-mediated regulation to microglial function; however, the exact underlying mechanism remains largely unknown. Here, we uncover the enrichment of miR-137, a neuropsychiatric-disorder-associated miRNA, in the microglial nucleus, and reveal its unexpected nuclear functions in maintaining the microglial global transcriptomic state, phagocytosis, and inflammatory response. Mechanistically, microglial Mir137 deletion increases chromatin accessibility, which contains binding motifs for the microglial master transcription factor Pu.1. Through biochemical and bioinformatics analyses, we propose that miR-137 modulates Pu.1-mediated gene expression by suppressing Pu.1 binding to chromatin. Importantly, we find that increased Pu.1 binding upregulates the target gene Jdp2 (Jun dimerization protein 2) and that knockdown of Jdp2 significantly suppresses the impaired phagocytosis and pro-inflammatory response in Mir137 knockout microglia. Collectively, our study provides evidence supporting the notion that nuclear miR-137 acts as a transcriptional modulator and that this microglia-specific function is essential for maintaining normal adult brain function.

The TET-Sall4-BMP regulatory axis controls craniofacial cartilage development.

Craniofacial microsomia (CFM) is a congenital defect that usually results from aberrant development of embryonic pharyngeal arches. However, the molecular basis of CFM pathogenesis is largely unknown. Here, we employ the zebrafish model to investigate mechanisms of CFM pathogenesis. In early embryos, tet2 and tet3 are essential for pharyngeal cartilage development. Single-cell RNA sequencing reveals that loss of Tet2/3 impairs chondrocyte differentiation due to insufficient BMP signaling. Moreover, biochemical and genetic evidence reveals that the sequence-specific 5mC/5hmC-binding protein, Sall4, binds the promoter of bmp4 to activate bmp4 expression and control pharyngeal cartilage development. Mechanistically, Sall4 directs co-phase separation of Tet2/3 with Sall4 to form condensates that mediate 5mC oxidation on the bmp4 promoter, thereby promoting bmp4 expression and enabling sufficient BMP signaling. These findings suggest the TET-BMP-Sall4 regulatory axis is critical for pharyngeal cartilage development. Collectively, our study provides insights into understanding craniofacial development and CFM pathogenesis.

Fragment-Based Deep Learning for Simultaneous Prediction of Polarizabilities and NMR Shieldings of Macromolecules and Their Aggregates.

Simultaneous prediction of the molecular response properties, such as polarizability and the NMR shielding constant, at a low computational cost is an unresolved issue. We propose to combine a linear-scaling generalized energy-based fragmentation (GEBF) method and deep learning (DL) with both molecular and atomic information-theoretic approach (ITA) quantities as effective descriptors. In GEBF, the total molecular polarizability can be assembled as a linear combination of the corresponding quantities calculated from a set of small embedded subsystems in GEBF. In the new GEBF-DL(ITA) protocol, one can predict subsystem polarizabilities based on the corresponding molecular wave function (thus electron density and ITA quantities) and DL model rather than calculate them from the computationally intensive coupled-perturbed Hartree-Fock or Kohn-Sham equations and finally obtain the total molecular polarizability via a linear combination equation. As a proof-of-concept application, we predict the molecular polarizabilities of large proteins and protein aggregates. GEBF-DL(ITA) is shown to be as accurate enough as GEBF, with mean absolute percentage error <1%. For the largest protein aggregate (>4000 atoms), GEBF-DL(ITA) gains a speedup ratio of 3 compared with GEBF. It is anticipated that when more advanced electronic structure methods are used, this advantage will be more appealing. Moreover, one can also predict the NMR chemical shieldings of proteins with reasonably good accuracy. Overall, the cost-efficient GEBF-DL(ITA) protocol should be a robust theoretical tool for simultaneously predicting polarizabilities and NMR shieldings of large systems.

PTK2B promotes TBK1 and STING oligomerization and enhances the STING-TBK1 signaling.

TANK-binding kinase 1 (TBK1) is a key kinase in regulating antiviral innate immune responses. While the oligomerization of TBK1 is critical for its full activation, the molecular mechanism of how TBK1 forms oligomers remains unclear. Here, we show that protein tyrosine kinase 2 beta (PTK2B) acts as a TBK1-interacting protein and regulates TBK1 oligomerization. Functional assays reveal that PTK2B depletion reduces antiviral signaling in mouse embryonic fibroblasts, macrophages and dendritic cells, and genetic experiments show that Ptk2b-deficient mice are more susceptible to viral infection than control mice. Mechanistically, we demonstrate that PTK2B directly phosphorylates residue Tyr591 of TBK1, which increases TBK1 oligomerization and activation. In addition, we find that PTK2B also interacts with the stimulator of interferon genes (STING) and can promote its oligomerization in a kinase-independent manner. Collectively, PTK2B enhances the oligomerization of TBK1 and STING via different mechanisms, subsequently regulating STING-TBK1 activation to ensure efficient antiviral innate immune responses.

Cdh1 plays a protective role in nonalcoholic fatty liver disease by regulating PPAR/PGC-1α signaling pathway.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a significant etiological factor in liver-related diseases, which can lead to severe consequences such as steatohepatitis, cirrhosis and death. Cdh1 is considered as a crucial protein involved in cell cycle regulation. The purpose of this study is to explore the biological role of Cdh1 in NAFLD. MATERIALS AND METHODS: NAFLD cell model was established, and L02 cells and AML12 cells were infected by shRNA lentivirus with Cdh1 knockdown in vitro, and the effect of Cdh1 deletion on cell lipid deposition was evaluated. The effects of Cdh1 deletion on Akt phosphorylation and PPAR/PGC-1α signaling pathway in L02 cells were examined. In addition, the NAFLD mouse model was constructed, and the conditional knockout mice of Cdh1 were selected to verify the results. RESULTS: In vitro experiments showed that the Cdh1 deletion enhanced cell lipid deposition. In vivo experiments showed that conditional knockdown of Cdh1 aggravated fatty degeneration and damage of liver in mice. Cdh1 deletion promotes Akt phosphorylation and inhibits PPAR/PGC-1α signaling pathway in L02 cells. Conditional knockout of Cdh1 down-regulates PPAR/PGC-1α signaling pathway in NAFLD mouse model. CONCLUSION: The deletion of Cdh1 may promote Akt phosphorylation by up-regulating Skp2 and inhibit the PPAR/PGC-1α signaling pathway. Cdh1 serves a protective function in the occurrence and progression of NAFLD.

The effect of synbiotics in patients with NAFLD: a systematic review and meta-analysis.

Background: Nonalcoholic fatty liver disease (NAFLD) is the highest incidence of chronic liver disease worldwide, seriously endangering human health, and its pathogenesis is still unclear. In the recent years, increasing evidence has shown that intestinal flora plays an important role in the occurrence and development of NAFLD. Synbiotics can alter gut microbiota and may be a treatment option for NAFLD in the future. Objectives: To systematically investigate the therapeutic effect of synbiotic supplementation on NAFLD patients. Design: A systematic review and meta-analysis were conducted. Data sources and methods: We conducted a search on four databases (PubMed, Embase, Cochrane Library, and Web of Science) to identify relevant studies. Eligible studies were then screened, and data from the included studies were extracted, combined, and analyzed. Result: This study analyzed 10 randomized controlled trials involving 634 patients with NAFLD. The results showed that synbiotic supplementation could significantly reduce the level of alanine aminotransferase (mean difference (MD) = -8.80; (95% CI [-13.06, -4.53]), p < 0.0001), aspartate aminotransferase (MD = -9.48; 95% CI [-12.54, -6.43], p < 0.0001), and γ-glutamyl transferase (MD = -12.55; 95% CI [-19.40, -5.69], p = 0.0003) in NAFLD patients. In the field of metabolism, synbiotic supplementation could significantly reduce the level of total cholesterol (MD = -11.93; 95% CI [-20.43, -3.42], p = 0.006) and low-density lipoprotein cholesterol (MD = -16.2; 95% CI [-19.79, -12.60], p < 0.0001) and increase the level of high-density lipoprotein cholesterol (MD = 1.56; 95% CI [0.43, 2.68], p = 0.007) in NAFLD patients. In addition, synbiotic supplementation could significantly reduce liver stiffness measurement indicator (MD = -1.09; 95% CI [-1.87, -0.30], p = 0.006) and controlled attenuation parameter indicator (MD = -37.04; 95% CI [-56.78, -17.30], p = 0.0002) in NAFLD patients. Conclusion: Based on the current evidence, synbiotic supplementation can improve liver function, adjust lipid metabolism, and reduce the degree of liver fibrosis in patients with NAFLD, but these effects need to be confirmed by further studies.

Identification of disease-related genes and construction of a gene co-expression database in non-alcoholic fatty liver disease.

Background: The mechanism of NAFLD progression remains incompletely understood. Current gene-centric analysis methods lack reproducibility in transcriptomic studies. Methods: A compendium of NAFLD tissue transcriptome datasets was analyzed. Gene co-expression modules were identified in the RNA-seq dataset GSE135251. Module genes were analyzed in the R gProfiler package for functional annotation. Module stability was assessed by sampling. Module reproducibility was analyzed by the ModulePreservation function in the WGCNA package. Analysis of variance (ANOVA) and Student's t-test was used to identify differential modules. The receiver operating characteristic (ROC) curve was used to illustrate the classification performance of modules. Connectivity Map was used to mine potential drugs for NAFLD treatment. Results: Sixteen gene co-expression modules were identified in NAFLD. These modules were associated with multiple functions such as nucleus, translation, transcription factors, vesicle, immune response, mitochondrion, collagen, and sterol biosynthesis. These modules were stable and reproducible in the other 10 datasets. Two modules were positively associated with steatosis and fibrosis and were differentially expressed between non-alcoholic steatohepatitis (NASH) and non-alcoholic fatty liver (NAFL). Three modules can efficiently separate control and NAFL. Four modules can separate NAFL and NASH. Two endoplasmic reticulum related modules were both upregulated in NAFL and NASH compared to normal control. Proportions of fibroblasts and M1 macrophages are positively correlated with fibrosis. Two hub genes Aebp1 and Fdft1 may play important roles in fibrosis and steatosis. m6A genes were strongly correlated with the expression of modules. Eight candidate drugs for NAFLD treatment were proposed. Finally, an easy-to-use NAFLD gene co-expression database was developed (available at https://nafld.shinyapps.io/shiny/). Conclusion: Two gene modules show good performance in stratifying NAFLD patients. The modules and hub genes may provide targets for disease treatment.

The association between sleep duration, quality, and nonalcoholic fatty liver disease: A cross-sectional study.

Sleep can affect nonalcoholic fatty liver disease (NAFLD). We investigated the association between sleep duration, sleep quality, and NAFLD. From January to December 2018, 1,073 patients (age: 37.94 ± 10.88, Body Mass Index (BMI): 22.85 ± 3.27) were enrolled. Pittsburgh Sleep Quality Index Questionnaire and Munich Chronotype Questionnaire were used to assess sleep duration, quality, and habits. Ultrasonography was used to diagnose NAFLD. Multivariate logistic regression models were used to calculate the odds ratio (OR) and 95% confidence interval (CI) of the risk of NAFLD by different types of sleep duration and sleep quality. No significant differences in sleep time, sleep quality, and sleep habits between the NAFLD and the non-NAFLD groups were observed (P > 0.05). There was no correlation between sleep duration and NAFLD in the whole cohort. After adjusting for age, exercise, fasting plasma glucose, and BMI, the group with long sleep duration showed a decreased risk of NAFLD in men (OR = 0.01, 95% CI: 0.001-0.27, P = 0.032). However, in all four adjusted models, no correlation between sleep duration, quality, and NAFLD was found in women. In conclusion, sleep duration was significantly and negatively associated with NAFLD in men but not women. Prospective studies are required to confirm this association.

RIG-I-mediated innate immune signaling in tumors reduces the therapeutic effect of oncolytic vesicular stomatitis virus.

BACKGROUND: Oncolytic viral therapy is a promising method for tumor treatment. Currently, several oncolytic viruses (OVs) have been used as tumor therapy at different phases of research and clinical trials. OVs not only directly lyse tumor cells due to viral replication but also initiate host antitumor immune responses. Previous studies have primarily focused on how OVs activate adaptive immune responses in immune cells. However, the role of innate immune responses in tumors induced by OVs remains unclear. METHODS: To determine the innate immune responses induced by vesicular stomatitis virus (VSV), the mutant VSVΔM51 strain was used for the infection and quantitative polymerase chain reaction (qPCR) was employed to measure the transcriptional levels of antiviral genes. The knockdown efficiency of RIG-I was examined by qPCR. Viral titers were measured by plaque assays. Tumor models were established by intradermally implanting RIG-I-knockdown and control LLC cells into the flank of wild type C57BL/6J mice. When the tumors reached approximately 50mm3 , they were infected with VSVΔM51 via intratumoral injections to examine its therapeutic effect. RESULTS: Infection with VSVΔM51 triggered remarkable innate immune responses in several tumor cell lines through the cytoplasmic RIG-I sensing pathway. Moreover, we found that intratumoral injection of VSVΔM51 effectively reduced tumor growth in murine LCC lung cancer model. Importantly, VSVΔM51 -induced antitumor therapy was more effective in murine LLC tumor model established using Rig-I-knockdown cells compared with the tumor model established using control cells. CONCLUSION: RIG-I-mediated innate immune signaling in tumor cells plays a negative role in regulating antitumor therapy with VSVΔM51 virus.

Argonaute-dependent ribosome-associated protein quality control.

Ribosome-associated protein quality control (RQC) is a protein surveillance mechanism that eliminates defective nascent polypeptides. The E3 ubiquitin ligase, Ltn1, is a key regulator of RQC that targets substrates for ubiquitination. Argonaute proteins (AGOs) are central players in miRNA-mediated gene silencing and have recently been shown to also regulate RQC by facilitating Ltn1. Therefore, AGOs directly coordinate post-transcriptional gene silencing and RQC, ensuring efficient gene silencing. We summarize the principles of RQC and the functions of AGOs in miRNA-mediated gene silencing, and discuss how AGOs associate with the endoplasmic reticulum (ER) to assist Ltn1 in controlling RQC. We highlight that RQC not only eliminates defective nascent polypeptides but also removes unwanted protein products when AGOs participate.

Pancancer analysis reveals the role of disulfidptosis in predicting prognosis, immune infiltration and immunotherapy response in tumors.

Disulfidptosis has been reported as a novel cell death process, suggesting a therapeutic strategy for cancer treatment. Herein, we constructed a multiomics data analysis to reveal the effects of disulfidptosis in tumors. Data for 33 kinds of tumors were downloaded from UCSC Xene, and disulfidptosis-related genes (DRGs) were selected from a previous study. After finishing processing data by the R packages, the expression and coexpression of DRGs in different tumors were assessed as well as copy number variations. The interaction network was drawn by STRING, and the activity of disulfidptosis was compared to the single-sample gene set enrichment analysis algorithm. Subsequently, the differences in DRGs for prognosis and clinicopathological features were evaluated, and the tumor immune microenvironment was assessed by the TIMER and TISCH databases. Tumor mutation burden, stem cell features and microsatellite instability were applied to predict drug resistance, and the expression of checkpoints was identified for the prediction of immunotherapy. Moreover, the TCIA, CellMiner and Enrichr databases were also utilized for selecting potential agents. Ten DRGs were differentially expressed in tumors, and the plots of coexpression and interaction revealed their correlation. Survival analysis suggested SLC7A11 as the most prognosis-related DRG with the most significant results. Additionally, the comparison also reflected the differences in DRGs in the status of pathologic lymph node metastasis for 5 types of tumors. The tumor immune microenvironment showed commonality among tumors based on immune infiltration and single-cell sequencing, and the analysis of tumor mutation burden, stemness and microsatellite instability showed a mostly positive correlation with DRGs. Moreover, referring to the prediction about clinical treatment, most DRGs can enhance sensitivity to chemotherapeutic agents but decrease the response to immune inhibitors with increasing expression. In this study, a primarily synthetic landscape of disulfidptosis in tumors was established and provided guidance for further exploration and investigation.

Ku proteins promote DNA binding and condensation of cyclic GMP-AMP synthase.

Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that plays a critical role in regulating antiviral signaling. cGAS binds to DNA and catalyzes the synthesis of cyclic GMP-AMP (cGAMP), which is essential for downstream signal transduction. The antiviral response is a rapid biological process; however, cGAS itself has relatively low DNA binding affinity, implying that formation of the cGAS-DNA complex requires an additional factor(s) that promotes cGAS-DNA binding, allowing efficient antiviral signal transduction. Here, we report that the Ku proteins (Ku80 and Ku70) directly interact with cGAS and positively regulate cGAS-mediated antiviral signaling. Mechanistically, we find that the interaction of the Ku proteins with cGAS significantly increases the DNA-binding affinity of cGAS and promotes cGAS condensation in the cytosol, thereby enhancing cGAS catalytic activity. Our results show that the Ku proteins are critical partners of cGAS in sensing DNA virus infection and ensuring efficient innate immune signal transduction.

A protocol for mimicking lipid-mediated phase separation on the membrane using giant unilamellar vesicles.

Here, we present a general protocol for mimicking lipid-mediated phase separation on the membrane using giant unilamellar vesicles (GUVs). In this protocol, we use GUVs to mimic Ago1 protein's phase separation behavior on the membrane through binding with phosphoinositides (PIPs). We provide procedures to prepare fluorescent-labeled Ago1 protein and PI(4,5)P2-containing GUVs, followed by steps to assess Ago1 protein's phase separation in 3D time-lapse images. This protocol can be applied to investigate a membrane-associated protein's behavior on the membrane. For complete details on the use and execution of this protocol, please refer to Gao et al. (2022).

A Density Functional Theory and Information-Theoretic Approach Study of Interaction Energy and Polarizability for Base Pairs and Peptides.

Using density functional theory (DFT) and the information-theoretic approach (ITA) quantities to appreciate the energetics and properties of biopolymers is still an unaccomplished and ongoing task. To this end, we studied the building blocks of nucleic acid base pairs and small peptides. For base pairs, we have dissected the relative importance of energetic components by using two energy partition schemes in DFT. Our results convincingly show that the exchange-correlation effect predominantly governs the molecular stability of base pairs while the electrostatic potential plays a minor but indispensable role, and the steric effect is trivial. Furthermore, we have revealed that simple density-based ITA functions are in good relationships with molecular polarizabilities for a series of 30 hydrogen-bonded base pairs and all 20 natural α-amino acids, 400 dipeptides, and 8000 tripeptides. Based on these lines, one can easily predict the molecular polarizabilities of larger peptides, even proteins as long as the total molecular wavefunction is available, rather than solving the computationally demanding coupled-perturbed Hartree-Fock (CPHF) equation or its DFT counterpart coupled-perturbed Kohn-Sham (CPKS) equation.

Optimized protocols for RNA-induced silencing complex assembly and cleavage in cultured Drosophila cells.

Here, we provide an optimized RNA-induced silencing complex (RISC) assembly and cleavage protocol in vitro without using radiolabeled RNA. The protocol is useful to characterize the biochemical properties of the RISC. We describe the preparation of RNA probes, the target RNA, and Drosophila cell lysates for RISC assembly assay. We then detail AGO1 complexes immunoprecipitation for RISC cleavage assay. This protocol can detect RISC assembly and cleavage products within 5 days. Moreover, it can detect 5'- and 3'-cleavage products simultaneously. For complete details on the use and execution of this protocol, please refer to Gao et al. (2022).