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The analysis of mRNA turnover often requires a knowledge of the pathway by which a particular mRNA is being degraded. In this article we describe experimental procedures that can be used to determine the mechanism of degradation for yeast transcripts. These approaches include the insertion of strong secondary structures to block exonuclease cleavage and thereby trap decay intermediates. In addition,...
mRNA decay is a multistep process, often dependent on the active translation of an mRNA and on components of the translation apparatus. InSaccharomyces cerevisiae,severaltrans-acting factors required for mRNA decay associate with polyribosomes. We have explored the specificity of the interactions of these factors with polyribosomes, using sucrose gradient sedimentation analysis of the yeastUPF1protein...
Modulation of mRNA stability provides a powerful means for controlling gene expression during the cell cycle, cell differentiation, the immune response, as well as many other physiological transitions. Through the years, many different methods have been developed for measuring mRNA stability. Frequently mRNA stability is studied indirectly by analyzing the steady-state level of mRNA. Therefore by...
Ribonucleases play essential roles in cell growth, differentiation, and the response to stress. This article deals with exoribonucleases, enzymes that degrade RNAs beginning at either the 5′ or 3′ end and proceed down the length of the RNA. The preparation of a crude extract of a mammalian 3′-to-5′ exonuclease is described. Assay conditions for both 5′-to-3′ and 3′-to-5′ exonucleases are given. One...
Endonucleases are key effectors of mRNA degradation, particularly for mRNAs whose turnover rates are regulated by extracellular stimuli. The rapid clearance of mRNA degradation productsin vivoand the need to selectively identify mRNA endonucleases in the presence of many other cellular ribonucleases make the study of these enzymes particularly challenging. We have successfully purified and cloned...
mRNA decapping is a common step shared between two important mRNA decay pathways in yeast,Saccharomyces cerevisiae.To investigate how mRNAs are decapped, we have developed an assay that can be easily used to measure the decapping activity. This assay has been used to isolate yeast strains with altered decapping activities. The results demonstrated that decreased decapping activityin vitrocorresponds...
The pathways and machinery involved in the regulated turnover of mRNAs in mammalian cells are largely unknown. We have developed anin vitrosystem using HeLa cytoplasmic S100 extracts and exogenous polyadenylated RNA substrates that faithfully reproducesin vivoaspects of regulated mRNA turnover. RNA substrates for use in the system that contain a poly(A) tail precisely at their 3′ end can be readily...
A + U-Rich elements (AREs) have been extensively investigated ascis-acting determinants of rapid mRNA turnover. Recently, a number of RNA-binding proteins interacting with AREs have been described. This article presents strategies and techniques used by our laboratory to identify and characterize a family of ARE-binding proteins collectively termed AUF1. However, these techniques may be applied to...
The poly(A) tail present at the 3′ end of most eukaryotic mRNAs can play a critical role in message translation and stability. Therefore, identifying alterations in poly(A) tail length can yield important insights into an mRNA's function and subsequent physiological impact. Here, we present three methods for assaying polyadenylation of a specific mRNA in the context of total cellular RNA. The first...
Regulation of mRNA turnover is a critical control mechanism of gene expression and is influenced by ribonucleoprotein (RNP) complexes that form onciselements. All mRNAs have an intrinsic half-life and in many cases these half-lives can be altered by a variety of stimuli that are manifested through the formation or disruption of an RNP structure. The stability of α-globin mRNA is determined by elements...
The analytical electron microscope technique called electron spectroscopic imaging (ESI) has a number of applications in the study of DNA:protein complexes. The method offers an intermediate level of spatial resolution forin vitrostructural studies of complexes that may be too large or heterogeneous to study by crystallography or magnetic resonance spectroscopy. An advantage of ESI is that the distribution...
DNA within a cell is organized with unrestrained torsional tension, and each molecule is divided into multiple individual topological domains. Psoralen photobinding can be used as an assay for supercoiling and topological domain size in living cells. Psoralen photobinds to DNA at a rate nearly linearly proportional to superhelical density. Comparison of the rate of photobinding to supercoiled and...
In the past decade, site-specific chromosomal DNA cleavage mediated by DNA endonucleases has been used to examine diverse aspects of chromosome structure and function in eukaryotes, such as DNA topology, replication, transcription, recombination, and repair. Here we describe a method with which chromosomes can be linearized at any predefined positionin vivo.Yeast homothallic switching endonuclease...
Methods are described for the utilization of formaldehyde as a reversible cross-linking agent for the characterization of protein–protein and protein–DNA interactions. The methods include a description of procedures to: (1) isolate and characterize transcriptionally active chromatin from cells cross-linked with formaldehyde; (2) study histone mobility during replication and transcription by the characterization...
Transcriptional regulation is a complex process that requires cooperation between specific DNA sequence elements, the DNA-binding proteins that bind to these sequences, the general transcriptional machinery, and chromatin. Oocyte microinjection offers a technique to study the integrated transcription process while still providing the opportunity to experimentally perturb this process. We describe...
A simple method for preparing chromatin assembly extracts has not been available for budding yeast. Here I describe such a method in detail. The assembly extract, a crude 100,000gsupernatant, is prepared from cells disrupted in a manual or motorized grinder while they are frozen. The core histones and all soluble protein factors required for chromatin assembly under physiological conditions are present...
Numerous regulatory mechanisms contribute to the control of eukaryotic transcription. These controls are manifested through higher-order protein–DNA structure within the nucleus.In vitroassays have proven extremely useful in deciphering the potential regulatory roles of chromatin and nuclear structure in transcription. Embryonic egg extracts ofXenopuswith their vast maternal stores and rapid cell-cycle...
We detail a method which allows biochemical quantities of histone proteins to be introduced into a living eukaryotic cell. This method involves absorption of purified proteins into macroplasmodia ofPhysarum polycephalum.Further, sincePhysarummacroplasmodia exist as syncitial culture with completely synchronous nuclei with respect to cell cycle events, proteins may be introduced at specific points...
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