MRNA stability

GPCR function has been shown to be regulated by several different mechanisms. The number of receptors on the plasma membrane may be regulated by transcription, mRNA stability, biosynthetic processing, and protein stability. In addition, the function of receptors in the plasma membrane can be influenced by regulatory phosphorylation and by association with other proteins that determine the subcellular location of receptors relative to other signaling molecules. 

The regulation of receptor synthesis is a second component of receptor downregulation. It involves processes that reduce gene transcription, mRNA stability, and receptor half-life time. It should be noted that mechanisms in addition to the regulation of the receptor number may account for tolerance development. Second messenger levels and enzyme activities that participate in the signaling of a given receptor are... 

Although most mRNAs in mammalian cells are very stable (half-lives measured in hours), some mrn over very rapidly (half-lives of 10-30 minutes). In certain instances, mRNA stability is subject to regulation. This has important implications since there is usually a direct relationship between mRNA amount and the translation of that mRNA into its cognate protein. Changes in the stability of a specific mRNA can therefore have major effects on biologic processes. 

Messenger RNAs exist in the cytoplasm as ribonu-cleoprotein particles (RNPs). Some of these proteins protect the mRNA from digestion by nucleases, while others may under certain conditions promote nuclease attack. It is thought that mRNAs are stabihzed or destabilized by the interaction of proteins with these various structures or sequences. Certain effectors, such as hormones, may regulate mRNA stability by increasing or decreasing the amount of these proteins. 

It appears that the ends of mRNA molecules are involved in mRNA stability (Figure 39-19). The 5 ... 

Figure 39-19. Structure of a typical eukaryotic mRNA showing elements that are involved in regulating mRNA stability. The typical eukaryotic mRNA has a 5 noncoding sequence (5 NCS), a coding region, and a 3 NCS. All are capped at the 5 end, and most have a polyadenylate sequence at the 3 end. The 5 cap and 3 poly(A) tail protect the mRNA against exonuclease attack. Stem-loop structures in the 5 and 3 NCS, features in the coding sequence, and the AU-rich region in the 3 NCS are thought to play roles in mRNA stability.

From the few examples cited, it is clear that a number of mechanisms are used to regulate mRNA stability—just as several mechanisms are used to regulate the synthesis of mRNA. Coordinate regulation of these two processes confers on the cell remarkable adaptability. 

Staton JM, Leedman PJ. Hormonal regulation of mRNA stability and RNA-pro-tein interactions in the pituitary. J Mol Endocrinol 2000 25 17-34. 

An example of miR-dependent deadenylation of mRNA measured by this method is shown in Fig. 6.3C. In this case, HeLa cells (which express let-7) were transfected with a pDNA R-luc construct encoding three let-7 binding sites in the 3 UTR (3 X bulge), or with control constructs encoding either no sites (plasmid) or three mutated let-7 sites (3 x bulge mut) (constructs described in Pillai et al, 2005). Cells were harvested 24 h after transfection, RNA was purified for the PAT assay, and luciferase activity was measured from cell lysates. As reported previously, the presence of functional let-7 target sites results in specific repression of luciferase expression with very minor effects on mRNA stability (Pillai et al., 2005). The experiment in Fig. 6.3C demonstrates that the let-7 targeted reporter mRNA is selectively deadenylated. 

Kedersha, N., and Anderson, P. (2002). Stress granules Sites of mRNA triage that regulate mRNA stability and translatability. Biochem. Soc. Trans. 30, 963—969. 

Hitti, E., Iakovleva, T., Brook, M., Deppenmeier, S., Gruber, A. D., Radzioch, D., Clark, A. R., Blackshear, P. J., Kotlyarov, A., and Gaestel, M. (2006). Mitogen-activated protein kinase-activated protein kinase 2 regulates tumor necrosis factor mRNA stability and translation mainly by altering tristetraprolin expression, stability, and binding to adenine/uridine-rich element. Mol. Cell Biol. 26, 2399—2407. 

The nature of the cap structure can also influence mRNA stability. The stability of mRNA capped with different ARCAs is determined in MM3MG cells that are electroporated with luciferase mRNAs containing a 60-nt poly(A) tract and different cap analogs. The experiment is done... 

Increased transcription levels are assumed to result in increased protein synthesis. One approach to reach this goal is to raise the transgene copy number by the use of amplification-promoting sequences derived from a spacer sequence of tobacco ribo-somal DNA [95]. Posttranscriptional processes such as capping, splicing and polya-denylation are important for high protein yields, and it is also important to maximize mRNA stability [84]. 

DON also enhances COX-2 mRNA stability.28 Such modulation is usually explained by the presence of multiple copies of AUUUA motif in the 3 -untranslated region (3 -UTR) of COX-2 mRNA, since the AU-rich element (ARE) in the 3 -UTR of an mRNA targets a transcript for rapid degradation.34 To test this possibility a constitutive SV40... 

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