Stability of mRNA

In order to examine the stabilities of mRNAs for the individual outer membrane proteins, the envelope fractions were separated by SDS polyacrylamide gel electrophoresis. From each time point, the rates of 

In contrast to stable mRNAs for the outer membrane proteins, mRNAs for the cytoplasmic membrane proteins were found to be as unstable as those for the cytoplasmic proteins. It is particularly interesting that only the mRNAs for the outer membrane proteins are stable and that there are differences in mRNA stabilities among the individual outer membrane proteins. It may be that these mRNAs are unusually stable be- 

Structural differences in mRNAs may cause not only different stabilities (half-lives) of mRNAs themselves, but may also cause a different affinity of mRNA to ribosomes. In the latter case, the mRNA to ribosomes affinity may result in a different stability of the initiation complex for protein synthesis, which possibly causes the different sensitivity to antibiotics known to be inhibitors of initiation of protein synthesis, such as kasugamycin. 

Osborn et al have shown that synthesis of lipopolysaccharide in S. typhimurium takes place exclusively in the cytoplasmic membrane. The specific pulse label (1 min at 25 C) of the polysaccharide chain (O-antigen) of the lipopolysaccharide initially appeared in the cytoplasmic membrane and was then rapidly transferred to the outer membrane during a subsequent chase. The mechanism of translocation of the completed lipopolysaccharide from the cytoplasmic membrane to the outer membrane is not yet clear. However, it has been shown by MUhlradt et al. that newly synthesized lipopolysaccharide is translocated at a few, localized sites on the S. typhimurium cell surface. These authors found that after a 30-sec pulse label with [ C]galactose, new lipopolysaccharide appeared at about 220 sites on the cell surface, and within 2-3 min, it was evenly distributed over the entire cell surface. When ultrathin sections of plasmolyzed cells were examined under the electron microscope after pulse labeling, newly synthesized lipopolysaccharide was found to be located above sites where the cytoplasmic and outer membranes were attached to each other. Such bridges between the two membranes may provide for the translocation of the newly synthesized lipopolysaccharide (see also Chapter 11). 

There is little doubt that the outer membrane proteins are synthesized either in the cytoplasm or on the inside surface of the cytoplasmic membrane. Therefore, as with lipopolysaccharide, the outer membrane proteins should also be transported from the cytoplasmic membrane to the outer membrane. It has not yet, however, been shown that the newly 

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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... 

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. 

In diabetes, it appears that stabilization of mRNA occurs, leading to an increased amount rather than an increase in synthesis. The induction caused by this disease may reflect the necessity to metabolize the ketone bodies produced. 

In most cases, the DNA inserted into the MCS for expression is not a genomic gene with the original exon/intron configuration, but a cDNA. In exceptional cases, antisense RNA or ribozymes may be expressed. cDNA lacks intronic sequences, but still has the 5 and 3 UTR sequences. As discussed above, the presence of the 3 UTR may reduce the stability of mRNA transcribed from the cDNA insert. Furthermore, the length and secondary structure of the 5 UTR may influence the efficiency of translation. Therefore, it is generally recommended to use cDNA with only short 5 and 3 UTR sequences for expression with expression vectors. 

Eukaryotic mRNAs often have long 3 untranslated sequences— sequences that follow the stop codon for the protein they encode. These mRNAs generally conclude with a sequence of up to 200 adenosines, the polyadenylic acid (polyA) sequence at the 3 end. This sequence isn t coded by the DNA template for the gene it is added post-transcriptionally. Not all mRNAs are polyadenylated. For example, histone mRNAs lack polyA tails. Polyadenylation seems to play a role in regulating the stability of mRNAs. An early event in the breakdown of some mRNAs is the removal of their polyA tails. 

If a large amount of protein is required over an extended period of time, this could be achieved by increasing the stability of mRNA. Gene amplification would be unnecessary. 

Non-receptor related mechanisms Enzyme induction may occur via non-receptor related mechanisms. CYP2A6, although known to be induced by phenobarbital and rifampin, may involve mRNA stabilization. CYP2E1 induction may be a result of stabilization of mRNA and protein. 

The role of the poly(A) tail is still not firmly established despite much effort. However, evidence that it enhances translation efficiency and the stability of mRNA is accumulating. Blocking the synthesis of the poly(A) tail by exposure... 

Anderson DM et al (2003) Stability of mRNA/cationic lipid lipoplexes in human and rat cerebrospinal fluid methods and evidence for nonviral mRNA gene delivery to the central nervous system. Hum Gene Ther 14 191-202... 

The role of the poly(A) tail is still not firmly established despite much effort. However, evidence is accumulating that it enhances translation elfi ciency and the stability of mRNA. Blocking the synthesis of the poly(A) tail by exposure to J deoxyadenosine (cordycepin) docs not interfere with iIk synthesis of the primary transcript. Messenger KNA devoid of a poly(A) tail can be transported out ol the nucleus, However, an mRNA molecule devoid of a poly(A) tail is usually a much less effective template tor protein synthesis than is one with a poly(A) tail. Indeed, some mRK As are stored in an unadenylated form and leceive the poly(A) tail only when translation is imminent. The half-life of an m RNA molecule may be determined in part by the rate of degradation of its poly(A ) tail. 

Figure 11-2 Complex response of human cells to sub-lethal levels of nitric oxide. IMR-90 cells (normal, diploid human embryonic lung fibroblasts) were exposed to NO at 280 nM s" for 90 minutes, and then pulse-labeled for a further 90 minutes prior to two-dimensional gel analysis. The protein species listed (by Mr) behaved reprodudbly over several experiments. At least two dififeient control pathways operate in this induction transcriptional activation, as seen with CL-100, a MAP kinase phosphatase, and stabilization of mRNA, as found for HO-1 (see [35] for details). The induction of HO-1 and other activities by NO confers adaptive resistance to NO in rodent motor neurons [40],...

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