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This volume provides new approaches and technologies into roles of poly(A) metabolism in translation, RNA stability, and quality control of gene expression. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls.
Authoritative and cutting-edge, Deadenylation: Methods and Protocols aims to pave the way for future investigations of the complex regulatory networks that control mRNA stability and expression.
List of contents
The reconstitution of human CCR4-NOT from purified proteins and an assay of its deadenylation activity.- In vitro reconstitution of the Drosophila melanogaster CCR4-NOT complex to assay deadenylation.- Analysis of human mRNA deadenylation complexes via high-resolution gel electrophoresis.- Quantitative biochemical analysis of deadenylase enzymes using fluorescence and chemiluminescence-based assays.- A FRET-based assay to quantify enzymatic rates and explore the mechanisms of RNA deadenylases in heterogeneous environments.- Measuring Poly-Adenosine Tail Length of RNAs by High Resolution Northern Blotting Coupled with RNase H Cleavage.- An RNA-ligation-based RACE-PAT assay to monitor poly(A) tail length of mRNAs of interest.- Dissecting the role of the Ccr4-Not deadenylase complex in pluripotency and differentiation.- Measuring proximity-mediated function of mRNA regulatory proteins by engineered tethering.-Tethered mRNA Amplifier: A NovelApproach to Increase Protein Expression.- RNA binding protein-mediated mRNA deadenylation in mammalian cell extracts.- Transcriptome-wide analysis of mRNA adenylation status in yeast using nanopore sequencing.- Sequencing of transcriptome-wide poly(A) tails by PAIso-seq.- Nano3'RACE: a method to analyze poly(A) tail length and nucleotide additions at the 3' extremity of selected mRNAs using nanopore sequencing.- Quantification of poly(A) tail length and terminal modifications using direct RNA sequencing.- Differential poly(A) tail length analysis using nanopore sequencing.- Single-molecule poly(A) tail sequencing (SM-PATseq) using the PacBio platform.- Mathematical models for the poly(A) tail shortening process.
