Stable RNAs

Table 35-1. Some of the species of small stable RNAs found in mammalian cells.

A large number of discrete, highly conserved, and small stable RNA species are found in eukaryotic cells. The majority of these molecules are complexed with proteins to form ribonucleoproteins and are distributed in the nucleus, in the cytoplasm, or in both. They range in... 

Cui Y, Rajasethupathy P, Hess GP (2004) Selecrion of stable RNA molecules that can regulate the channel-opening equilibrium of the membrane-bound gamma-aminobutyric acid receptor. Biochemistry 43 16442-16449... 

As we shall see in the next chapter, some natural RNA molecules catalyze the formation of peptide bonds, offering an idea of how the RNA world might have been transformed by the greater catalytic potential of proteins. The synthesis of proteins would have been a major event in the evolution of the RNA world, but would also have hastened its demise. The informationcarrying role of RNA may have passed to DNA because DNA is chemically more stable. RNA replicase and reverse transcriptase may be modem versions of enzymes that once played important roles in making the transition to the modern DNA-based system. 

Since RNA secondary structure is relatively stable, RNA folding experiments which start with a sample that has been prepared in the absence of Mg2 1 generally need to anneal the sample in a suitable buffer at relatively high temperatures between 50 and 90 °C. 

There are three major classes of RNA in cells messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). Of these, the latter two are termed stable RNAs, as they have a longer half-life than that of mRNA [1], Ribosomal RNA is the most abundant class of RNA in a cell. In a typical eukaryotic cell (yeast, plant, and animal), there are other RNAs, such as organelle RNA and small RNAs in nuclei (snRNAs) or in the cytoplasm (7S RNA). In eukaryotic cells, most RNAs are synthesized as larger precursor molecules and are then processed into smaller mature RNAs. Total RNA in a human cell may range from 10 to 30 pg, with most of it in the cytoplasm (about 85%), while the rest is in the nucleus. 

Unlike DNA, RNA is very susceptible to rapid degradation due to ribonu-cleases (RNases), which are highly stable RNA degrading enzymes. In addition, RNA is more labile than DNA, especially at higher temperatures (>65°C) and at alkaline pH (>9). The sensitivity of RNA toward alkaline hydrolysis can be used for selective hydrolysis of RNA in a mixture of RNA and DNA [5], Isolation of intact RNA is crucial to the success of many applications, such as the measurement of qualitative and quantitative changes in gene expression, preparation of cDNA or cDNA libraries, and in the synthesis of a probe for various molecular hybridization experiments. 

Other stable RNAs in the cell are involved in protein and RNA processing. Processing means changes in a protein after the synthesis of the peptide bond, or in an RNA after the synthesis of phosphodiester bonds during transcription. Some of these RNAs are catalytic. 

Eukaryotic transcription uses three distinct RNA polymerases, which are specialized for different RNAs. RNA polymerase I makes Ribosomal RNAs, RNA polymerase II makes messenger RNAs, and RNA polymerase III makes small, stable RNAs such as transfer RNAs and 5S ribosomal RNA. Eukaryotic RNA polymerases are... 

Macromolecular biomass composition is of obvious interest when the biomass itself is the product, such as algal biomass in [18], or for production of singlecell protein, for e.g. animal feedstock. Moreover, for a precise metabolic flux analysis, changes in biomass composition should be taken in account. For example, Henriksen et al. [19] observed with E. coli under different growth rates, that the levels of DNA and lipids were relatively constant, whereas the proteins and stable RNA levels increased with the specific growth rate and the total amount of carbohydrates decreased. 

Alluvia S, Weinstein-Eischer D, Zhang A, Postow L, Storz G. A small, stable RNA induced by oxidative stress Role as a pleiotropic regulator and antimutator. Cell 1997 90(1) 43—53. 

The 3 ends of transcripts are frequently ragged and map to one sequence region or to closely spaced regions. For a few of the stable RNA genes the termination site mapped to... 

Transcripts of 5S rRNAs and tRNAs generally start just before and terminate immediately after single genes amongst sulfothermophiles, while these stable RNA genes are often clustered in transcriptional units in the thermophilic methanogens and other archaea. 

The function of many noncoding RNAs (ncRNAs) depend on a defined secondary structure. RNAz detects evolutionarily conserved and thermodynamically stable RNA secondary structures in multiple sequence alignments and, thus, efficiently filters for candidate ncRNAs. In this chapter, we provide a step-by-step guide on how to use RNAz. Starting with basic concepts, we also cover advanced analysis techniques and, as an example for a large scale application, demonstrate a complete screen of the Saccharomyces cerevisiae genome. 

Hofacker, I. L., Priwitzer, B., and Stadler, P. F. (2004) Prediction of locally stable RNA secondary structures for genome-wide surveys. Bioinformatics 20, 186-190. 

RNA polymerase I synthesizes only pre-rRNA. RNA polymerase II synthesizes mRNAs and some of the small nuclear RNAs that participate in mRNA splicing. RNA polymerase III synthesizes tRNAs, 5S rRNA, and several other relatively short, stable RNAs. 

Transcription initiation by Pol 1, which synthesizes pre-rRNA, and by Pol 111, which synthesizes tRNAs, 5S rRNA, and other short, stable RNAs, has been characterized most extensively In S. cerevisiae using both biochemical and genetic approaches. It is clear that synthesis of tRNAs and of rRNAs, which are incorporated Into ribosomes, is tightly coupled to the rate of cell growth and proliferation. However, much remains to be learned about how transcription initiation by Pol I and Pol III is regulated so that synthesis of pre-rRNA, 5S rRNA, and tRNAs Is coordinated with the growth and replication of cells. 

Imamura, T., Kanai, F., Kawakami, T., Amarsanaa, J., Ijichi, H., Hos-hida, Y, et al. (2004). Proteomic analysis of the TGF-beta signaling pathway in pancreatic carcinoma cells using stable RNA interference to silence Smad4 expression. Biochemical and Biophysical Research Communications, 318, 289—296. 

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