往期刊物2023
卷册: 13, 期号: 18
生物化学
Computational Analysis of Plasma Lipidomics from Mice Fed Standard Chow and Ketogenic Diet
标准饲料和生酮饮食小鼠血浆脂质组学的计算分析
Dietary saturated fatty acids (SFAs) are upregulated in the blood circulation following digestion. A variety of circulating lipid species have been implicated in metabolic and inflammatory diseases; however, due to the extreme variability in serum or plasma lipid concentrations found in human studies, established reference ranges are still lacking, in addition to lipid specificity and diagnostic biomarkers. Mass spectrometry is widely used for identification of lipid species in the plasma, and there are many differences in sample extraction methods within the literature. We used ultra-high performance liquid chromatography (UPLC) coupled to a high-resolution hybrid triple quadrupole-time-of-flight (QToF) mass spectrometry (MS) to compare relative peak abundance of specific lipid species within the following lipid classes: free fatty acids (FFAs), triglycerides (TAGs), phosphatidylcholines (PCs), and sphingolipids (SGs), in the plasma of mice fed a standard chow (SC; low in SFAs) or ketogenic diet (KD; high in SFAs) for two weeks. In this protocol, we used Principal Component Analysis (PCA) and R to visualize how individual mice clustered together according to their diet, and we found that KD-fed mice displayed unique blood profiles for many lipid species identified within each lipid class compared to SC-fed mice. We conclude that two weeks of KD feeding is sufficient to significantly alter circulating lipids, with PCs being the most altered lipid class, followed by SGs, TAGs, and FFAs, including palmitic acid (PA) and PA-saturated lipids. This protocol is needed to advance knowledge on the impact that SFA-enriched diets have on concentrations of specific lipids in the blood that are known to be associated with metabolic and inflammatory diseases.Key features• Analysis of relative plasma lipid concentrations from mice on different diets using R.• Lipidomics data collected via ultra-high performance liquid chromatography (UPLC) coupled to a high-resolution hybrid triple quadrupole-time-of-flight (QToF) mass spectrometry (MS).• Allows for a comprehensive comparison of diet-dependent plasma lipid profiles, including a variety of specific lipid species within several different lipid classes.• Accumulation of certain free fatty acids, phosphatidylcholines, triglycerides, and sphingolipids are associated with metabolic and inflammatory diseases, and plasma concentrations may be clinically useful.Graphical overview
Fractionation of Native Protein Complexes from Mammalian Cells to Determine the Differential Proteasome Activity and Abundance
分离哺乳动物细胞中的天然蛋白复合物以测定差异蛋白酶体活性和丰度
Eukaryotic cells have different types of proteasomes that differ in size. The smallest proteolytically active particle is the 20S proteasome, which degrades damaged and oxidized proteins; the most common larger particle is the 26S proteasome, which degrades ubiquitylated proteins. The 26S proteasome is formed by a 20S particle capped with one or two regulatory particles, named 19S. While proteasome particles function in the cytoplasm, endoplasmic reticulum, and nucleus, our understanding of their abundance and activity in different cellular compartments is still limited. We provide a three-step protocol that first involves detergent-based fractionation of the cytoplasmic and nuclear compartments, maintaining the integrity and activity of proteasome complexes. Second, the protocol employs native gel separation of large multiprotein complexes in the fractions and a fluorescence-based in-gel quantitation of the activity and different proteasome particles. Finally, the protocol involves protein in-gel denaturation and transfer to a PVDF membrane. Western blotting then detects and quantifies the different proteasome particles. Therefore, the protocol allows for sensitive measurements of activity and abundance of individual proteasome particles from different cellular compartments. It has been optimized for motor neurons induced from mouse embryonic stem cells but can be applied to a variety of mammalian cell lines.Key features• Protocol for fractionation of active nuclear and cytoplasmic proteasome complexes.• Native electrophoresis and fluorescence-based in-gel activity assay, which allows the visualization and quantification of active complexes within the acrylamide gel matrix.• In-gel protein denaturation followed by transfer of complexes to PVDF membrane, which allows the analysis of complexes’ abundance using antibodies.Graphical overview
Identification of Matriglycan by Dual Exoglycosidase Digestion of α-Dystroglycan
通过双外糖苷酶消化 α-肌聚糖来鉴定基质聚糖
Matriglycan is a linear polysaccharide of alternating xylose and glucuronic acid units [-Xyl-α1,3-GlcA-β1,3]n that is uniquely synthesized on α-dystroglycan (α-DG) and is essential for neuromuscular function and brain development. It binds several extracellular matrix proteins that contain laminin-globular domains and is a receptor for Old World arenaviruses such as Lassa Fever virus. Monoclonal antibodies such as IIH6 are commonly used to detect matriglycan on α-DG. However, endogenous expression levels are not sufficient to detect and analyze matriglycan by mass spectrometry approaches. Thus, there is a growing need to independently confirm the presence of matriglycan on α-DG and possibly other proteins. We used an enzymatic approach to detect matriglycan, which involved digesting it with two thermophilic exoglycosidases: β-Glucuronidase from Thermotoga maritima and α-xylosidase from Sulfolobus solfataricus. This allowed us to identify and categorize matriglycan on α-DG by studying post-digestion changes in the molecular weight of α-DG using SDS-PAGE followed by western blotting with anti-matriglycan antibodies, anti-core α-DG antibodies, and/or laminin binding assay. In some tissues, matriglycan is capped by a sulfate group, which renders it resistant to digestion by these dual exoglycosidases. Thus, this method can be used to determine the capping status of matriglycan. To date, matriglycan has only been identified on vertebrate α-DG. We anticipate that this method will facilitate the discovery of matriglycan on α-DG in other species and possibly on other proteins.Key features• Analysis of endogenous matriglycan on dystroglycan from any animal tissue.• Matriglycan is digested using thermophilic enzymes, which require optimum thermophilic conditions.• Western blotting is used to assay the success and extent of digestion.• Freshly purified enzymes work best to digest matriglycan.Graphical overview α-Dystroglycan (α-DG) from muscle is shown here modified by a phosphorylated core M3 glycan, which extends further and terminates in a repeating disaccharide of xylose (Xyl) and glucuronic acid (GlcA) called matriglycan. β-glucuronidase (Bgus) and α-xylosidase (Xyls) hydrolyze the β-1,3-linked GlcA and α-1,3 linked-Xyl, starting from the terminal residues.
生物工程
Quantitative Analysis of Clot Deposition on Extracorporeal Life Support Membrane Oxygenators Using Digital and Scanning Electron Microscopy Imaging Techniques
使用数字和扫描电子显微镜成像技术对体外生命支持膜氧合器上的凝块沉积进行定量分析
Device-induced thrombosis remains a major complication of extracorporeal life support (ECLS). To more thoroughly understand how blood components interact with the artificial surfaces of ECLS circuit components, assessment of clot deposition on these surfaces following clinical use is urgently needed. Scanning electron microscopy (SEM), which produces high-resolution images at nanoscale level, allows visualization and characterization of thrombotic deposits on ECLS circuitry. However, methodologies to increase the quantifiability of SEM analysis of ECLS circuit components have yet to be applied clinically. To address these issues, we developed a protocol to quantify clot deposition on ECLS membrane oxygenator gas transfer fiber sheets through digital and SEM imaging techniques. In this study, ECLS membrane oxygenator fiber sheets were obtained, fixed, and imaged after use. Following a standardized process, the percentage of clot deposition on both digital images and SEM images was quantified using ImageJ through blind reviews. The interrater reliability of quantitative analysis among reviewers was evaluated. Although this protocol focused on the analysis of ECLS membrane oxygenators, it is also adaptable to other components of the ECLS circuits such as catheters and tubing.Key features• Quantitative analysis of clot deposition using digital and scanning electron microscopy (SEM) techniques• High-resolution images at nanoscale level• Extracorporeal life support (ECLS) devices• Membrane oxygenators• Blood-contacting surfacesGraphical overview
生物科学
Intraperitoneal Injection of Neonatal Mice
新生小鼠腹腔注射
Administration of substances into neonatal mice is required for early treatment with pre-clinical therapeutics, delivery of recombination-inducing substances, and dosing with viruses or toxins, amongst other things. Several injection routes into mouse pups are possible, including intravenous and intracerebroventricular, each with their own advantages and limitations. Here, we describe a simple and rapid protocol for the intraperitoneal injection of neonatal mice for systemic dosing. By detaching a 30-gauge needle from its plastic hub and inserting it into polyethylene tubing attached to a Hamilton syringe, small volumes (1–10 μL) can be accurately injected into the peritoneal cavity of pups aged 1–5 days old. The procedure can be completed within a few minutes, is generally safe and well tolerated by both pups and parents, and can be used in combination with alternative administration routes.Key features• This protocol provides a simple description to rapidly and efficiently inject mouse pups aged 1–5 days for systemic dosing.• Allows treatment of neonatal mice with substances such as viruses and compounds for research across disciplines.Graphical overview
细胞生物学
Isolation of Epithelial and Stromal Cells from Colon Tissues in Homeostasis and Under Inflammatory Conditions
在稳态和炎症条件下从结肠组织中分离上皮细胞和基质细胞
Inflammation of the gastrointestinal tract is a prevalent pathology in diseases such as inflammatory bowel disease (IBD). Currently, there are no therapies to prevent IBD, and available therapies to treat IBD are often sub-optimal. Thus, an unmet need exists to better understand the molecular mechanisms underlying intestinal tissue responses to damage and regeneration. The recent development of single-cell RNA (sc-RNA) sequencing-based techniques offers a unique opportunity to shed light on novel signaling pathways and cellular states that govern tissue adaptation or maladaptation across a broad spectrum of diseases. These approaches require the isolation of high-quality cells from tissues for downstream transcriptomic analyses. In the context of intestinal biology, there is a lack of protocols that ensure the isolation of epithelial and non-epithelial compartments simultaneously with high-quality yield. Here, we report two protocols for the isolation of epithelial and stromal cells from mouse and human colon tissues under inflammatory conditions. Specifically, we tested the feasibility of the protocols in a mouse model of dextran sodium sulfate (DSS)-induced colitis and in human biopsies from Crohn’s patients. We performed sc-RNA sequencing analysis and demonstrated that the protocol preserves most of the epithelial and stromal cell types found in the colon. Moreover, the protocol is suitable for immunofluorescence staining of surface markers for epithelial, stromal, and immune cell lineages for flow cytometry analyses. This optimized protocol will provide a new resource for scientists to study complex tissues such as the colon in the context of tissue damage and regeneration.Key features• This protocol allows the isolation of epithelial and stromal cells from colon tissues.• The protocol has been optimized for tissues under inflammatory conditions with compromised cell viability.• This protocol is suitable for experimental mouse models of colon inflammation and human biopsies.Graphical overviewGraphical representation of the main steps for the processing of colon tissue from dextran sodium sulfate (DSS)-treated mice (upper panel) and frozen biopsies from Crohn’s patients (lower panel)
免疫学
Functional Phenotyping of Lung Mouse CD4+ T Cells Using Multiparametric Flow Cytometry Analysis
使用多参数流式细胞术分析小鼠肺 CD4+ T 细胞的功能表型
Gammaherpesviruses such as Epstein-Barr virus (EBV) are major modulators of the immune responses of their hosts. In the related study (PMID: 35857578), we investigated the role for Ly6Chi monocytes in shaping the function of effector CD4+ T cells in the context of a murine gammaherpesvirus infection (Murid gammaherpesvirus 4) as a model of human EBV. In order to unravel the polyfunctional properties of CD4+ T-cell subsets, we used multiparametric flow cytometry to perform intracellular staining on lung cells. As such, we have developed herein an intracellular staining workflow to identify on the same samples the cytotoxic and/or regulatory properties of CD4+ lymphocytes at the single-cell level. Briefly, following perfusion, collection, digestion, and filtration of the lung to obtain a single-cell suspension, lung cells were cultured for 4 h with protein transport inhibitors and specific stimulation media to accumulate cytokines of interest and/or cytotoxic granules. After multicolor surface labeling, fixation, and mild permeabilization, lung cells were stained for intracytoplasmic antigens and analyzed with a Fortessa 4-laser cytometer. This method of quantifying cytotoxic mediators as well as pro- or anti-inflammatory cytokines by flow cytometry has allowed us to decipher at high resolution the functional heterogeneity of lung CD4+ T cells recruited after a viral infection. Therefore, this analysis provided a better understanding of the importance of CD4+ T-cell regulation to prevent the development of virus-induced immunopathologies in the lung.Key features• High-resolution profiling of the functional properties of lung-infiltrating CD4+ T cells after viral infection using conventional multiparametric flow cytometry.• Detailed protocol for mouse lung dissection, preparation of single-cell suspension, and setup of multicolor surface/intracellular staining.• Summary of optimal ex vivo restimulation conditions for investigating the functional polarization and cytokine production of lung-infiltrating CD4+ T cells.• Comprehensive compilation of necessary biological and technical controls to ensure reliable data analysis and interpretation.Graphical overviewGraphical abstract depicting the interactions between immune cells infiltrating the alveolar niche and the lung during respiratory infection with a gammaherpesvirus (Murid herpesvirus 4, MuHV-4). Two distinct situations are represented: the inflammatory response developed during viral replication in the lung, either in the presence (WT mice) or absence of regulatory monocytes (CCR2KO mice). Sequential process of the experiment is represented, starting from intratracheal instillation of MuHV-4 virions to tissue dissociation and multicolor staining for flow cytometry analysis.
Differentiation of Bone Marrow Monocytes into Alveolar Macrophages-like Cells through Co-culture with Lung Epithelial Cells and Group 2 Innate Lymphoid Cells
通过与肺上皮细胞和第 2 组先天淋巴细胞共培养将骨髓单核细胞分化为肺泡巨噬细胞样细胞
During life, the embryonic alveolar macrophage (AM) population undergoes successive waves of depletion and replenishment in response to infectious and inflammatory episodes. While resident AMs are traditionally described as from embryonic origin, their ontogeny following inflammation or infection is much more complex. Indeed, it appears that the contribution of monocytes (MOs) to the AM pool is variable and depends on the type of inflammation, its severity, and the signals released in the microenvironment of the pulmonary niche (peripheral imprinting) and/or in the bone marrow (central imprinting). Deciphering the cellular and molecular mechanisms regulating the differentiation of MOs into AMs remains an area of intense investigation, as this could potentially explain part of the inter-individual susceptibility to respiratory immunopathologies. Here, we detail a relevant ex vivo co-culture model to investigate how lung epithelial cells (ECs) and group 2 lung innate lymphoid cells (ILC2s) contribute to the differentiation of recruited MOs into AMs. Interestingly, the presence of lung ILC2s and ECs provides the necessary niche signals to ensure the differentiation of bone marrow MOs into AMs, thus establishing an accessible model to study the underlying mechanisms following different infection or inflammation processes.Key features• Ex vivo co-culture model of the alveolar niche.• Deciphering the particular niche signals underlying the differentiation of MO into AMs and their functional polarization.Graphical overviewThis protocol described the isolation of bone marrow monocytes (MOs), lung epithelial cells (ECs), and lung group 2 lung innate lymphoid cells (ILC2s) and the ex vivo co-culture of these cells to drive the differentiation of bone marrow MOs into alveolar macrophages (AMs).This co-culture experiment is composed of three steps (Graphical overview):1. Identification and FACS-sorting of ECs and MOs isolated from the lung and the bone marrow of naive mice, respectively. 2. Culture of these ECs and bone marrow MOs for three days.3. Addition of ILC2s isolated from the lung of naïve mice or mice subjected to a treatment/infection of interest.
Double Staining with Fluorescent Tracers to Determine Myeloid Cell Migration of Leishmania-infected Cells from Mouse Skin to Lymphatic Tissues by Flow Cytometry
用荧光示踪剂双重染色通过流式细胞术测定利什曼原虫感染细胞从小鼠皮肤到淋巴组织的骨髓细胞迁移
Immune cell trafficking in steady-state conditions and inflammatory cell recruitment into injured tissues is crucial for the surveillance of the immune system and the maintenance of body homeostasis. Tracking the cell journey from the infection site in the skin to lymphoid tissues has been challenging, and is typically determined using fluorescent cell tracers, antibodies, or photoconvertible models. Here, we describe the detailed method to track Leishmania-infected myeloid cells migrating from the skin to lymphatic tissues by multiparametric flow cytometry. These methods involve labeling of infective Leishmania donovani parasites with fluorescent cell tracers and phenotyping of myeloid cells with fluorescent antibodies, to determine the infection status of migratory myeloid cells. We also describe the detailed protocol to trace donor monocytes transferred intradermally into recipient mice in Leishmania donovani infection. These protocols can be adapted to study skin-lymphoid tissue migration of dendritic cells, inflammatory monocytes, neutrophils, and other phagocytic myeloid cells in response to vaccine antigens and infection.Key features• Cell-tracking of cell-trace-labeled parasites and monocytes from the skin to lymphatic tissues after transference into donor mice.• Identification of migratory cells labeled with fluorescent cell tracers and antibodies by flow cytometry.• Isolation, labeling, and transference of bone marrow monocytes from donor mice into the skin of recipient mice.• Description of a double-staining technique with fluorescent cell tracers to determine cell and parasite dissemination from the skin to lymphoid tissues.Graphical overviewOverview of the methods to trace the migration of Leishmania and monocytes from the skin to lymphatic tissues by flow cytometry. Infective metacyclic promastigotes (from axenic culture) and monocytes (isolated from the bone marrow of donor mice) are labeled with fluorescent cell tracers. After intradermal injection into the test mouse (1, 2), migratory cells and infected cells are isolated from the skin and lymphoid tissues of the test mouse. These cells are then labeled with fluorescent antibodies against myeloid cells and recognized according to the differential excitation/emission wavelengths of the fluorochromes by flow cytometry.
分子生物学
Controlled Level of Contamination Coupled to Deep Sequencing (CoLoC-seq) Probes the Global Localisation Topology of Organelle Transcriptomes
控制污染水平与深度测序 (CoLoC-seq) 相结合以探索细胞器转录组的全局定位拓扑
Information on RNA localisation is essential for understanding physiological and pathological processes, such as gene expression, cell reprogramming, host–pathogen interactions, and signalling pathways involving RNA transactions at the level of membrane-less or membrane-bounded organelles and extracellular vesicles. In many cases, it is important to assess the topology of RNA localisation, i.e., to distinguish the transcripts encapsulated within an organelle of interest from those merely attached to its surface. This allows establishing which RNAs can, in principle, engage in local molecular interactions and which are prevented from interacting by membranes or other physical barriers. The most widely used techniques interrogating RNA localisation topology are based on the treatment of isolated organelles with RNases with subsequent identification of the surviving transcripts by northern blotting, qRT-PCR, or RNA-seq. However, this approach produces incoherent results and many false positives. Here, we describe Controlled Level of Contamination coupled to deep sequencing (CoLoC-seq), a more refined subcellular transcriptomics approach that overcomes these pitfalls. CoLoC-seq starts by the purification of organelles of interest. They are then either left intact or lysed and subjected to a gradient of RNase concentrations to produce unique RNA degradation dynamics profiles, which can be monitored by northern blotting or RNA-seq. Through straightforward mathematical modelling, CoLoC-seq distinguishes true membrane-enveloped transcripts from degradable and non-degradable contaminants of any abundance. The method has been implemented in the mitochondria of HEK293 cells, where it outperformed alternative subcellular transcriptomics approaches. It is applicable to other membrane-bounded organelles, e.g., plastids, single-membrane organelles of the vesicular system, extracellular vesicles, or viral particles.Key features• Tested on human mitochondria; potentially applicable to cell cultures, non-model organisms, extracellular vesicles, enveloped viruses, tissues; does not require genetic manipulations or highly pure organelles.• In the case of human cells, the required amount of starting material is ~2,500 cm2 of 80% confluent cells (or ~3 × 108 HEK293 cells).• CoLoC-seq implements a special RNA-seq strategy to selectively capture intact transcripts, which requires RNases generating 5′-hydroxyl and 2′/3′-phosphate termini (e.g., RNase A, RNase I).• Relies on nonlinear regression software with customisable exponential functions.Graphical overview
A Post-translational Modification–enhanced Pull-down Method to Study Degron Domains and the Associated Protein Degradation Complexes
一种翻译后修饰增强的牵引法，用于研究 Degron 结构域和相关的蛋白质降解复合物
The identification and characterization of the ubiquitin E-ligase complexes involved in specific proteins’ degradation via the ubiquitin-proteasome system (UPS) can be challenging and require biochemical purification processes and in vitro reconstitution assays. Likewise, evaluating the effect of parallel phosphorylation and ubiquitination events occurring in vivo at dual phospho/ubiquitin-regulated motifs (called Phospho-Degrons or pDegrons) driving UPS degradation of the targeted protein has remained elusive. Indeed, the functional study of such E1-E2-E3 complexes acting on a protein-specific level requires previously or otherwise acquired knowledge of the nature of such degradation complex components. Furthermore, the molecular basis of the interaction between an E3 ligase and its pDegron binding motif on a target protein would require individually optimized in vitro kinase and ubiquitination assays. Here, we describe a novel enzymatically enhanced pull-down method to functionally streamline the discovery and functional validation of the ubiquitin E-ligase components interacting with a phospho-degron containing protein domain and/or sub-domain. The protocol combines key features of a protein kinase and ubiquitination in vitro assay by including them in a pull-down step exerted by a known or putative pDegron-tagged peptide using the cell extracts as a source of enzymatically active post-translational modification (PTM) modifying/binding native proteins. The same method allows studying specific stimuli or treatments towards the recruitment of the molecular degradation complex at the target protein’s phospho-degron site, reflecting in vivo–initiated events further enhanced through the assay design. In order to take full advantage of the method over traditional protein–protein interaction methods, we propose to use this PTM-enhanced (PTMe) pull down both towards the degradation complex discovery/ID phase as well as for the functional pDegron recruitment validation phase, which is the one described in the present protocol both graphically and in a stepwise fashion for reproduceable results.Key features• Suitable to study UPS-regulated (a) cytosolic and/or nuclear proteins, (b) intracellular region of transmembrane proteins, and (c) protein sub-domains bearing a known/putative pDegron motif.• Requires a biotin-tagged recombinant version of the target protein and/or sub-domain.• Allows the qualitative and quantitative analysis of endogenous ubiquitin (Ub) E-ligases recruitment to a known or putative pDegron bearing protein/sub-domain.• Allows simultaneous testing of various treatments and/or conditions affecting the phosphorylative and/or ubiquitylation status of the studied pDegron bearing protein/sub-domain and the recruited factors.Graphical overview
Immunoprecipitation of Reporter Nascent Chains from Active Ribosomes to Study Translation Efficiency
活性核糖体报告新生链的免疫沉淀研究翻译效率
The study of translation is important to the understanding of gene expression. While genome-wide measurements of translation efficiency (TE) rely upon ribosome profiling, classical approaches to address translation of individual genes of interest rely on biochemical methods, such as polysome fractionation and immunoprecipitation (IP) of ribosomal components, or on reporter constructs, such as luciferase reporters. Methods to investigate translation have been developed that, however, require considerable research effort, including addition of numerous features to mRNA regions, genomic integration of reporters, and complex data analysis. Here, we describe a simple biochemical reporter assay to study TE of mRNAs expressed from a transiently transfected plasmid, which we term Nascent Chain Immunoprecipitation (NC IP). The assay is based on a plasmid expressing an N-terminally Flag-tagged protein and relies on the IP of Flag-tagged nascent chains from elongating ribosomes, followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) quantification of eluted mRNA. We report that elution of mRNA following IP can be achieved by treatment with puromycin, which releases ribosome-mRNA complexes, or with purified Flag peptide, which instead releases nascent chain-ribosome-mRNA complexes. In the example described in this protocol, untranslated regions (UTRs) of a gene of interest were used to flank a FlagVenus coding sequence, with the method allowing to infer UTR-dependent regulation of TE. Importantly, our method enables discrimination of translating from non-translating mRNAs. Additionally, it requires simple procedures and standard laboratory equipment. Our method can be used to test the effect of regulators, such as microRNAs or therapeutic drugs or of various genetic backgrounds, on translation of any user-selected mRNA.Key features• The novel NC IP protocol builds upon a previously published method for detection of mRNA-binding proteins (Williams et al., 2022).• The NC IP protocol is adapted for detecting mRNA actively undergoing translation.• The method uses mammalian cell culture but could be adapted to multiple organisms, including budding yeast (S. cerevisiae).Graphical overview Design of the Nascent Chain Immunoprecipitation (NC IP) reporter and assay. Left. The construct carries a 3× Flag tag at the N-terminal end of Venus protein (FlagVenus). In this example, the reporter is adapted to study untranslated regions (UTR)-dependent expression by flanking FlagVenus coding sequence with UTRs of Aurora kinase A (AURKA) mRNA (depicted reporters refer to Cacioppo et al., 2023, Figure 3). The depicted reporters carry mutations in the proximal (p) or distal (d) polyadenylation signal (PAS). Right. Following reporter transfection, ribosomes are locked onto reporter mRNA by treating cells with cycloheximide (CHX), which prevents ribosome run-off and additional rounds of elongation, before cell lysis and immunoprecipitation (IP) of FlagVenus nascent chains via anti-Flag beads. Reporter mRNAs are then eluted, isolated, and quantified by RT-qPCR.
Successful Transfection of MicroRNA Mimics or Inhibitors in a Regular Cell Line and in Primary Cells Derived from Patients with Rheumatoid Arthritis
在常规细胞系和源自类风湿性关节炎患者的原代细胞中成功转染 MicroRNA 模拟物或抑制剂
The transfection of microRNA (miRNA) mimics and inhibitors can lead to the gain and loss of intracellular miRNA function, helping us better understand the role of miRNA during gene expression regulation under specific physical conditions. Our previous research has confirmed the efficiency and convenience of using liposomes to transfect miRNA mimics or inhibitors. This work uses miR-424 as an example, to provide a detailed introduction for the transfection process of miRNA mimics and inhibitors in the regular SW982 cell line and primary rheumatoid arthritis synovial fibroblasts (RASF) cells from patients by using lipofection, which can also serve as a reference to miRNA transfection in other cell lines.Key features• MiRNA mimics and inhibitors transfection in regular SW982 cell line and primary RASF cells.• Treatment and culture of RASF primary cells before transfection.Using liposomes for transfection purposes.
Fluorescence in situ Localization of Pri-miRNAs in Isolated Arabidopsis thaliana Nuclei
拟南芥离体细胞核中 Pri-miRNA 的荧光原位定位
Here, we present an approach combining fluorescence in situ hybridization (FISH) and immunolabeling for localization of pri-miRNAs in isolated nuclei of A. thaliana. The presented method utilizes specific DNA oligonucleotide probes, modified by addition of digoxigenin-labeled deoxynucleotides to its 3′ hydroxyl terminus by terminal deoxynucleotidyl transferase (TdT). The probes are then detected by immunolabeling of digoxigenin (DIG) using specific fluorescent-labeled antibodies to visualize hybridized probes. Recently, we have applied this method to localize pri-miRNA156a, pri-miRNA163, pri-miRNA393a, and pri-miRNA414 in the nuclei isolated from leaves of 4-week-old A. thaliana. The present approach can be easily implemented to analyze nuclear distribution of diverse RNA classes, including mRNAs and pri-miRNAs in isolated fixed cells or nuclei from plant.
Genome-wide Mapping of 5′-monophosphorylated Ends of Mammalian Nascent RNA Transcripts
哺乳动物新生 RNA 转录本 5′-单磷酸化末端的全基因组定位
In eukaryotic cells, RNA biogenesis generally requires processing of the nascent transcript as it is being synthesized by RNA polymerase. These processing events include endonucleolytic cleavage, exonucleolytic trimming, and splicing of the growing nascent transcript. Endonucleolytic cleavage events that generate an exposed 5′-monophosphorylated (5′-PO4) end on the growing nascent transcript occur in the maturation of rRNAs, tRNAs, and mRNAs. These 5′-PO4 ends can be a target of further processing or be subjected to 5′-3′ exonucleolytic digestion that may result in termination of transcription. Here, we describe how to identify 5′-PO4 ends of intermediates in nascent RNA metabolism. We capture these species via metabolic labeling with bromouridine followed by immunoprecipitation and specific ligation of 5′-PO4 RNA ends with the 3′-hydroxyl group of a 5′ adaptor (5′-PO4 Bru-Seq) using RNA ligase I. These ligation events are localized at single nucleotide resolution via highthroughput sequencing, which identifies the position of 5′-PO4 groups precisely. This protocol successfully detects the 5′monophosphorylated ends of RNA processing intermediates during production of mature ribosomal, transfer, and micro RNAs. When combined with inhibition of the nuclear 5′-3′ exonuclease Xrn2, 5′-PO4 Bru-Seq maps the 5′ splice sites of debranched introns and mRNA and tRNA 3′ end processing sites cleaved by CPSF73 and RNaseZ, respectively.Key features• Metabolic labeling for brief periods with bromouridine focuses the analysis of 5′-PO4 RNA ends on the population of nascent transcripts that are actively transcribed.• Detects 5′-PO4 RNA ends on nascent transcripts produced by all RNA polymerases.• Detects 5′-PO4 RNA ends at single nucleotide resolution.
