Cellular RNA

Wang, J., and Baltimore, D. (1983). Cellular RNA homologous to the Abelson murine leukemia virus transforming gene expression and relationship to the viral sequence. Mol. Cell. Biol. 3 773-779. 

Most eukaryotic mRNA molecules have up to 250 adenine bases at their 3 end. These poly (A) tails can be used in the affinity chromatographic purification of mRNA from a total cellular RNA extract. Under high salt conditions, poly (A) will hybridize to oligo-dT-cellulose or poly(U)-sepharose. These materials are polymers of 10 to 20 deoxythymidine or uridine nucleotides covalently bound to a carbohydrate support. They bind mRNA containing poly (A) tails as short as 20 residues. rRNA and tRNA do not possess poly (A) sequences and will not bind. After washing the mRNA can be eluted with a low salt buffer. 

Figure 2 Cytokine gene expression in immunopotentiating reconstituted influenza virosomes (IRIV) stimulated peripheral blood mononuclear cells (PBMC). PBMC were cultured in the presence or absence of IRIV. On days 1 and 2, culture cells were harvested and total cellular RNA was extracted and reverse transcribed. The cDNAs thus obtained were tested in real time polymerase chain reaction assays in the presence of primers specific for the indicated cytokine genes. Source From Refs. 6 and 9.

Brahic, M. and Ozden, S. (1992) Simnltaneous detection of cellular RNA and proteins, in In situ hybridization, Oxford University Press, Oxford, UK, pp. 85-102. 

Cellular RNAs vary widely in their size, structure, and lifespan. The great majority of them are ribosomal RNA (rRNA), which in several forms is a structural and functional component of ribosomes (see p.250). Ribosomal RNA is produced from DNA by transcription in the nucleolus, and it is processed there and assembled with proteins to form ribosome subunits (see pp.208, 242). The bacterial 16S-rRNA shown in Fig. A, with 1542 nucleotides (nt), is a component of the small ribosomae subunit, while the much smaller 5S-rRNA (118 nt) is located in the large subunit. 

DNA is a linear polymer of deoxyribonucleotides in which the sequence of purine and pyrimidine bases encodes cellular RNA and protein molecules. 

Most types of cellular RNA are involved in various steps in protein synthesis or gene expression. 

Cells have many ribosomes, so rRNAs comprise the majority (- 80%) of cellular RNA. 

Additionally, the cellular location at which the resultant polypeptide will function often cannot be predicted from RNA detection/sequences nor can detailed information regarding how the polypeptide product s functional activity will be regulated (e.g. via post-translational mechanisms such as phosphorylation, partial proteolysis, etc.). Therefore, protein-based drug leads/targets are often more successfully identified by direct examination of the expressed protein complement of the cell, i.e. its proteome. Like the transcriptome (total cellular RNA content) and in contrast to the genome, the proteome is not static with changes in cellular... 

In all procedures prior to hybridization, utmost precautions must be taken to prevent the contamination of ribonucleases, which results in the degradation of cellular RNA. Gloves must be worn throughout the steps. PBS and water must be autoclaved and, if possible, should be treated with DEPC (trrNote 4). All containers must be clean and ribonuclease-free. Particularly, it is important that, if reused, 12-well plates and insert buckets are treated with HjOj and DEPC (rrrNote 5). 

Messenger RNA is only one of several classes of cellular RNA. Transfer RNAs serve as adapter molecules in protein synthesis covalently linked to an amino acid at one end, they pair with the mRNA in such a way that amino acids are joined to a growing polypeptide in the correct sequence. Ribosomal RNAs are components of ribosomes. There is also a wide variety of special-function RNAs, including some (called ribozymes) that have enzymatic activity. All the RNAs are considered in detail in Chapter 26. The diverse and often complex functions of these RNAs reflect a diversity of structure much richer than that observed in DNA molecules. 

Probes may also consist of DNA copied from mRNA. This is known as cDNA and is also widely used to determine indirectly the sequences of mRNA molecules. Messenger RNA may be isolated from the total cellular RNA by affinity chromatography on bound poly (dT) or poly (U). These materials selectively hold RNA with the poly (A) tails characteristic of most eukaryotic mRNA (see Chapter 28). Another source of mRNA is polyribosomes (polysomes), which are "reading" mRNA and actively making proteins. 

In this chapter we focus on the structure and metabolism of the major classes of cellular RNA. 

The bulk of the cellular RNA is ribosomal RNA. Although seven genes exist in E. coli for rRNA, they all lead to essentially the same three ribosomal RNA molecules (see table 28.1) which differ substantially in size. The three rRNAs are always found in a complex with proteins in a functional component known as the ribosome. The ribosome is the site where mRNA and tRNAs meet to engage in protein synthesis. In E. coli, ribosomes are referred to as 70S particles, a measure of their rate of sedimentation and hence their size (S refers to Svedberg units, which are defined in chapter 6). A 70S ribosome consists of two dissociable subunits A 50S subunit and a 30S subunit. Each of these contains both RNA and protein. The 50S subunit contains 23S and 5S rRNAs. The 30S subunit contains a single 16S rRNA (fig. 28.5). Eukaryotic ribosomes are similar in structure, although they are somewhat larger (80S) and con-... 

Fischer, U., Huber, J., Boelens, W.C., Mattaj, I.W. and Luhrmann, R. (1995) The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell, 82, 475 183. 

The methods of RNA isolation depends on the tissue and type of RNA to be extracted. Procedures to isolate total cellular RNA include chemical extractions and centrifugation. mRNA is isolated from total RNA using affinity chromatography or magnetic beads, while high-pressure liquid chromatography methods are used for small RNA molecules. Phenol extraction was one of the first techniques to isolate RNA successfully from many sources... 

Northern blotting follows much the same procedure as Southern blotting except that the sample analyzed by gel electrophoresis and then bound to the filter is RNA not DNA. Therefore the technique detects RNA molecules that are complementary to the DNA probe. If cellular RNA is electrophoresed, for example, a DNA probe for a specific mRNA could be used to detect whether that mRNA was present in the sample. The migration distance of the RNA in the gel would also allow estimation of its size. Note that Southern blotting (for DNA) obtained its name after its inventor (E. Southern) the name Northern blotting (for RNA) was devised later and is a geographical pun ... 

Besides being incorporated into proteins, methionine is the source of methyl groups for several important reactions, including the modification of cellular RNAs and the biosynthesis of lipids. 

---