Stability of the mRNA

The concentration of mRNA available for translation is determined by the de novo synthesis, as well as by the degradation rate. The specific degradation of mRNAs plays an important role in cell- and tissue-specific gene expression (review Sachs, 1993). The stabUity of various mRNAs can vary significantly from 20 min to 24 hour half-life in the same cell. 

The following discussion gives examples of control elements important for the stability and degradation of mRNA. 

Tubulin is a well-studied example of the interference of gene products with the stability of mRNA. Processed tubulin binds as a dimer to the growing tubulin chain on the ribosome. The binding of the tubulin facilitates the attack by endonucleases on the ribo-some-boimd tubulin mRNA and thereby initiates the degradation of the mRNA (Fig. 1.52). The goal of this regulation process is to prevent the formation of excess tubulin. If tubulin is in excess, then the degradation of its own mRNA is induced and the synthesis of more tubulin is prevented. 

Two proteins are important for iron metabolism in mammalian cells the transferrin receptor (TFR) md ferritin. Ferritin is a protein for the storage of iron. The production and its level is increased when more iron is available. 

TFR is instrumental in the uptake of iron imder conditions of low iron concentration. The concentration of cellular TFR is inversely correlated with the level of iron if little iron is present, then the TFR concentration is increased if high levels of iron are present, then the TFR concentration is decreased. 

Several decay pathways exist for mRNAs. The major type of decay pathway is dependent on the deadenylation of mRNAS another pathway is independent of it. Furthermore, a pathway has been identified that targets mRNAS with nonsense codons and degrades aberrant RNAs. 

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Regulation of expression may occur at both the transcriptional and post-transcriptional levels. The mRNA for GM-CSF contains (in common with those of some other cytokines) conserved regulatory sequences in the 3 untranslated region, which may affect its rate of translation. The gene is constitutively transcribed in monocytes, endothelial cells and fibroblasts, but the mRNA is unstable and so does not accumulate to levels sufficient to allow translation into significant amounts of protein. Activation of these cells results in the increased expression of GM-CSF protein, which arises from both an enhanced rate of transcription (as detected in nuclear runoff experiments) and also an increased stability of the mRNA, perhaps by mechanisms analogous to those described above during activation of G-CSF expression ( 2.2.3.1). 

An extremely important role of iron is the synthesis of haem for formation of erythrocytes and also for proliferating cells for synthesis of the mitochondrial enzymes that contain haem (e.g. cytochromes). The flux-generating enzyme in the synthesis of haem is aminolevulinic acid synthase (ALS) (Figure 15.20). If the cellular iron concentration is low, the concentration of this enzyme is increased in an attempt to maintain the rate of synthesis. As with the other two proteins, the concentration of ALS is controlled at the level of translation in a similar manner to that for transferrin, i.e. by increased stability of the mRNA, which is achieved by the binding of the IRP to the mRNA. 

Hie regulation of the ferritin concentration is also related to the iron concentration. Hie vulnerable point is not the stability of the mRNA, but rather of the initiation of translation. Hie mRNA for ferritin possesses a hairpin structure in the 5 - non-coding... 

A second type of mechanism is illustrated by the malic enzyme and fatty synthetase systems. In such systems the effect of T3 on the accumulation of the specific mRNAs does not seem to depend only on direct gene activation by the T3-nuclear receptor. An amplification post-transcriptional mechanism seems to contribute to the accumulation of the specific mRNAs. Stabilization of the mRNA level is also differently modulated by other hormones and by the diet. 

The stability of the mRNA transcripts is another determinant of translational efficiency. It has been postulated that mRNAs are subjected to alternative decay processes based on their structural characteristics and the kinetics of these decay processes vary across time-scales [22], By incorporating selected stabilizing structural elements within the mRNA transcripts, one could, in theory, improve mRNA lifetime. By coupling this phenomenon to other structural-based properties such as translational throughput and re-initiation of translation, one could effectively tune gene expression to optimize throughput of a pathway. 

Fig. 1. Transgenic constructs. A map of the transgene constructs used to generate the various lines of mice that resulted in endogenous expression of an EGFP-tagged a - AR. To drive endogenous expression, 3.4- and 4.4-kb mouse promoter fragments were used for the a1B- and a1A-AR, respectively. The EGFP, a fluorescent protein, was placed inframe after the stop codon of the wild-type human a1B-AR and a1A-AR cDNAs. SV40 polyA sequence was used to increase stability of the mRNA.

Fig. 1.45 Regulation of the stability of the mRNA ofthe transferrin receptor by Fe3+. The translation of the mRNA ofthe transferrin receptor (TFR) is subject to regulation by the Fe concentration. Fe exerts its regulatory effect via the iron regulatory protein typel (IRP1). The IRP1 binds to a control segment at the 3 terminal region ofthe TFR mRNA, known as the iron responsive element (IRE). Binding of IRP1 to a hairpin structure of the IRE stabilizes the mRNA ofthe transferrin receptor and...

Like many of the Aux/IAA mRNAs, SAURs accumulate in response to cycloheximide treatment [61]. The response with soybean SAURs does not, however, result from derepression of transcription, but appears to result exclusively from stabilization of the mRNAs. SAURs represent some of the most unstable mRNAs identified in plants [62,63]. 

Aside from the 5 and 3 ends, some internal modihcations of the mRNA such as a phosphorothiate backbone or T modifications of residues can be brought into the mRNA as long as the modified residues are substrate for the RNA polymerases routinely used for in vitro transcription (T7, T3, or SP6 RNA polymerases) and do not interfere with the translation process. It seems that only a few modifications such as phosphorothioate nucleotides and 2 amino residues can be used [14]. However, they lower the efficacy of transcription and translation while they do not noticeably increase the stability of the mRNA toward RNases. Some more work may allow the identifications of modifications that may allow the efficacious production of mRNA, which resists extracellular RNases and remains well translatable by the ribosome. Such mRNA would be optimized compared with native mRNA for therapeutic uses. 

The function of the nuclear encoded PSI subunits is still unknown. Mutants affected in the synthesis of these subunits could provide valuable insights into this problem. We have therefore screened 58 PSI mutants by Northern hybridization using the cDNAs of P21, P28, P30, P35 and P37 as probes. No significant change in the transcript levels of any of these subunits was found. A similar approach with nuclear PSII mutants has identified mutations affecting the transcription and/or stabilization of the mRNAs of the PSII OEEl and 0EE2 proteins (17,18). One possibility is that the absence of one of these nuclear encoded PSI subunits still allows for partial PSI activity. Mutants of this sort would be leaky in contrast to the mutants screened which were all completely deficient in PSI activity. Another possibility is that some of the mutants are affected at the translational level. Antibodies against the individual polypeptides are required to test this hypothesis. 

Dozin, B., Quarto, R., Rossi, F., and Cancedda, R., 1990, Stabilization of the mRNA follows transcriptional activation of type II collagen gene in differentiating chicken chondrocyte, J. Biol. Chem. 265 7216-7220. 

The influence of actinomycin on the differentiation of erythroidal cells in the yolk sac of mouse embryos has been investigated. It was found that up to the 11th day of embryogenesis, a nonhemoglobin protein is synthesized. This phase is sensitive to actinomycin. Thus, a short living mRNA is responsible for the synthesis of this protein. After day 11, hemoglobin is synthesized. This synthesis is not sensitive to actinomycin treatment. The synthesis is, rather, associated with the stabilization of the mRNA template which controls hemoglobin molecule synthesis (Marx and Kovach, 1966 Wilt, 1967 Ryabov and Shostka, 1973). 

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