RNA Turnover in Eukaryotes: Nucleases, Pathways and Analysis of mRNA Decay

Specific complexes of protein and RNA carry out many essential biological functions, including RNA processing, RNA turnover, RNA folding, as well as the translation of genetic information from mRNA into protein sequences. Messenger RNA (mRNA) decay is now emerging as an important control point and a major contributor to gene expression. Continuing identification of the protein factors and cofactors, and mRNA instability elements responsible for mRNA decay allow researchers to build a comprehensive picture of the highly orchestrated processes involved in mRNA decay and its regulation.

Covers the nonsense-mediated mRNA decay (NMD) or mRNA surveillance pathwayExpert researchers introduce the most advanced technologies and techniques to identify mRNA processing, transport, localization and turnover, which are central to the process of gene expressionOffers step-by-step lab instructions, including necessary equipment and reagents

Three-hybrid analysis to study RNA decay
S. cerevisiae (i.e, G18 insertions, mutations in nucleases…)
Defining degradative activities in Drosophila melanogaster
Defining degradative activities in plants
RT-PCR product sequencing of mRNA decay intermediates in mammalian cells after decapping and circularization in vitro
Tethering Assays
Assays of poly(A) tail length (PAT assay, specific oligo-dT anchors…) and polarity of mRNA decay (Invader Assay)
In vivo crosslinking-coupled affinity purification of mRNAs for decay analysis (Streptavidin, biotin…)
Classification of ribonucleases by computational analyses
Analysis of P bodies in Saccharomyces cerevisiae
Analysis of stress granules and P bodies in mammalian cells, including neurons PMR1-mediated mRNA decay
No-go mRNA in Saccharomyces cerevisiae
mRNA decay mediated by decay factor adaptors (SLBP, PUF, Staufen1…)
Non-stop decay in Saccharomyces cerevisiae
Measuring ARE-mediated mRNA decay in cells
Measuring ARE-mediated mRNA decay in animals
Methods to study viral-mediated influences on mRNA decay
Measuring Saccharomyces cerevisiae mitochondrial mRNA half-life
Measuring mRNA decay in Arabidopsis mitochondria
Measuring mRNA decay in human mitochondria
Measuring mRNA decay in chloroplasts
• Nonsense-Mediated mRNA Decay (how do you study NMD, what defines an
In Saccharomyces cerevisiae
In Drosophila melanogaster
In Caenorhabditis elegans
In plants
In mammals
• Analysis of Nuclear RNA Decay
Measuring nuclear RNA decay in Saccharomyces cerevisiae (includes pre-mRNA, mRNA, rRNA, TRAMP complex…)
Measuring nuclear RNA decay in mammals
• tRNA Decay
Decay of improperly modified tRNA decay in Sacharomyces cerevisiae
• rRNA Decay
Decay of nonfunctional rRNA in Sacharomyces cerevisiae
• RNAi-Mediated mRNA Decay
Defining RNAi targets (e.g., Ago knockdowns, identifying complementary siRNA/miRNA…)
Studying miRNAs in unicellular organisms (Chlamydamones reinhardtii) Studying miRNAs in unicellular organisms: RNAi-dependent mRNA turnover in fission yeast:
Assays for microRNA-mediated mRNA decay in Caenhorabditis elegans
Analyses of microRNA function in zebra fish
Targeted deletion of cell-type specific microRNAs in mice for developmental studies