Titlebook: RNA-Protein Complexes and Interactions; Methods and Protocol Ren-Jang Lin Book 2023Latest edition The Editor(s) (if applicable) and The Aut

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Global Assessment of Protein Translation in Mammalian Cells Using Polysome Fractionation,ellular translation activity and can be assessed after biochemical fractionations of polysomes. Polysome fractionation begins with immobilizing ribosomes on mRNAs using inhibitors of translation elongation, for example, cycloheximide. Nuclei-free cell lysates are then isolated and layered on the top

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,Fluorescent In Situ Detection of RNA–Protein Interactions in Intact Cells by RNA-PLA,ation of a circular DNA template occurs if the target RNA and protein are within 40 nanometers of each other. The resulting circular template is amplified by rolling circle amplification and abundantly recognized by fluorescent antisense DNA oligonucleotides. This strategy therefore enables localiza

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Arresting Spliceosome Intermediates at Various Stages of the Splicing Pathway,tand the molecular mechanism of the splicing reaction, it is necessary to dissect the spliceosome pathway and isolate spliceosome intermediates in various stages of the pathway for biochemical and structural analysis. Here, we describe protocols for preparing intron-containing transcripts, cell-free

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,Streamlined Purification of RNA–Protein Complexes Using UV Cross-Linking and RNA Antisense Purificadescribe an updated capture method to identify direct and specific RNA–protein interactions. First, RNA and protein are covalently cross-linked in living cells by treatment with UV light at 254 nanometers wavelength. The antisense purification approach is dependent upon nucleic acid hybridization be

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,MS2-MBP-Based Affinity Purification of Nucleus- or Cytoplasm-Localized lncRNA–Protein Complexes Forthe role of lncRNA and its biological function. However, lncRNP purification is still a daunting challenge. Here we describe a protocol to purify lncRNP formed in vivo with MS2-MBP-based affinity purification. Inducible lncRNA tagged with MS2 RNA hairpins is introduced into cells of interest, and RN

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RNA and Protein Interactomes of an RNA-Binding Protein Tagged with FLAG Epitopes Using Combinatory cellular functions, the complete knockout of these proteins may be lethal to the cell. Overexpression of RBPs, on the other hand, may create an altered transcriptome and aberrant phenotypes that can mask their physiological function. Additionally, biochemical characterization of RBP often requires

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Detecting R-Loop Formation Using a Plasmid-Based In Vitro Transcription Assay,y Ronald Davis and colleagues over 40 years ago, the study of R-loops has become an increasingly expanded area of research. Numerous factors have been identified to modulate the dynamic formation and resolution of R-loops, which are critical for proper controls of gene expression and genome stabilit

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Electrophoretic Mobility Shift Assay (EMSA) and Microscale Thermophoresis (MST) Methods to Measure ious tRNAs toward the homologs of Elp3 from various organisms. The rather qualitative results from EMSA analyses can be nicely complemented by MST measurements allowing precise determination of the dissociation constant (K.). We also provide detailed notes for users to mitigate potential ambiguities and technical pitfalls during the procedures.

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Chemical Probing of RNA Structure In Vivo Using SHAPE-MaP and DMS-MaP, interaction network. In this chapter, we describe methods for characterizing the in vivo nucleotide flexibility of RNA in cells by SHAPE-MaP (SHAPE by .ut.tional .rofiling) and DMS-MaP. In another chapter, we will discuss the characterization of RNA-protein interaction network by RNP-MaP.