No abstract

The abundance of a cytoplasmic mRNA in eukaryotes often determines the level of the encoded protein product. The rates at which an mRNA is synthesized, exported, and degraded collectively contribute to its abundance in all cell types. Numerous mRNAs, particularly those encoding structural proteins, are very stable, with half-lives in the order of many hours. In contrast, mRNAs encoding regulatory proteins, including oncoproteins, cytokines, and signaling proteins, are relatively unstable with half-lives of an hour or less. As a result, modest changes in their decay rates affect their levels over a relatively short time period. This is particularly important to ensure rapid responses to extracellular signaling events. Messenger RNAs often harbor sequence elements that dictate their degradation rates. Adenylate uridylate (A+U)-rich elements (AREs), first identified in 1986, are perhaps the best characterized sequences that promote rapid mRNA degradation. These elements, localized within 3′-untranslated regions, sometimes contain AUUUA pentamers within an overall U-rich sequence, but this does not always define a bona fide ARE. Thus, experimental validation is essential before bestowing upon a suspected A+U-rich sequence the title of "ARE." This chapter describes a reporter gene system that permits quantitative assessment of the effects of candidate A+U-rich sequences on mRNA half-life. This system employs tetracycline-controlled transcriptional silencing of the reporter gene, isolation of total-cell RNA at selected time points, quantitative reverse transcriptase polymerase chain reaction analysis of reporter mRNA levels, and nonlinear regression analysis of mRNA level as a function of time to quantitatively define parameters describing mRNA decay kinetics. Finally, this chapter describes more specialized assays to characterize ARE-mediated mRNA decay pathways, including deadenylation, and discusses decapping.

RNA binding proteins are a large and varied group of factors that are the driving force behind post-transcriptional gene regulation. By analogy with transcription factors, RNA binding proteins bind to various regions of the mRNAs that they regulate, usually upstream or downstream from the coding region, and modulate one of the five major processes in mRNA metabolism: splicing, polyadenylation, export, translation and decay. The most abundant RNA binding protein domain is called the RNA Recognition Motif (RRM) 1 . It is probably safe to say that an RRM-containing protein is making some contact with an mRNA throughout its existence. The transcriptional counterpart would likely be the histones, yet the multitude of specific functions that are results of RRM based interactions belies the universality of the motif. This complex and diverse application of a single protein motif was used as the basis to develop an advanced graduate level seminar course in RNA:protein interactions. The course, utilizing a learner-centered empowerment model, was developed to dissect each step in RNA metabolism from the perspective of an RRM containing protein. This provided a framework to discuss the development of specificity for the RRM for each required process.Keywords: RNA recognition motif, splicing, RNA export, translation, polyadenylation, mRNA localization, mRNA decay.A wide range of proteins are able to associate with mRNA via highly conserved RNA-binding domains and play a pivotal role in mRNA metabolism and hence, in the post-transcriptional regulation of gene expression. The RNA recognition motif (RRM), alternatively is one of the most abundant eukaryotic protein domains that mediate interaction between mRNAs and proteins. The pFAM database currently lists over 12,000 sequences containing RRM motifs derived from over 400 species [1]. In humans there are 500 RRM-containing proteins annotated, suggesting that 2% of the genome is dedicated to RRM production. The observation that precursor mRNAs and nuclear mRNAs are almost always found complexed with proteins led to the initial detection of RRMs 20 years ago [2]. Since then, RRM containing proteins have been extensively studied in conjunction with their function in the development of RNA and gene expression [3][4][5]. The RRM comprises a consensus RNAbinding sequence about 75-85 amino acids long, located at N-termini of proteins, the structure of which is a b 1 a 1 b 2 b 3 a 2 b 4 fold [6]. RNA-binding protein motifs are functionally conserved between eukaryotes and prokaryotes and often bind other proteins in addition to RNA [7]. Here we review the function of several RRM-containing proteins that are crucial to the process of RNA metabolism from transcription through decay highlighting specific proteins involved at each stage. Figure 1 illustrates the life-cycle of an mRNA making note of the RRM-containing proteins discussed below. mRNA SPLICINGThe spliceosome is an intricate organization of protein/ RNA complexes whose primary purpose is to remove introns from pre-mRNA. Accur...

Regulation of expression of HTLV-1 gene products from integrated proviruses plays an important role in HTLV-1-associated disease pathogenesis. Previous studies have shown that T cell receptor (TCR)- and phorbol ester (PMA) stimulation of chronically infected CD4 T cells increases the expression of integrated HTLV-1 proviruses in latently infected cells, however the mechanism remains unknown. Analysis of HTLV-1 RNA and protein species following PMA treatment of the latently HTLV-1-infected, FS and SP T cell lines demonstrated rapid induction of tax/rex mRNA. This rapid increase in tax/rex mRNA was associated with markedly enhanced tax/rex mRNA stability while the stability of unspliced or singly spliced HTLV-1 RNAs did not increase. Tax/rex mRNA in the HTLV-1 constitutively expressing cell lines exhibited high basal stability even without PMA treatment. Our data support a model whereby T cell activation leads to increased HTLV-1 gene expression at least in part through increased tax/rex mRNA stability.