Eukaryotic mRNAs purification

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. 

The base pairings between A-T(U) and G-C are exploited in the purification of nucleic acids, such as purification of specific genes with immobilized oligonucleotides. An interesting example of applying hybridization between DNA and mRNA is the purification of eukaryotic mRNA (having a polyA sequence at 3 ends) with polyU affinity chromatography. 

The presence of the poly A sequence has been exploited for the isolation and purification of eukaryotic mRNA, by using affinity columns of oligo-dT-cellulose. 

Poly(uridylic acid) is firmly adsorbed by cellulose, furnishing a derivative that was used in the purification of eukaryotic mRNAs. ... 

Walden WE, Daniels McQueen S, Brown PH, Gaffield L, Russell DA, Bielser D, Bailey LD, Thach RE (1988) Translational repression in eukaryotes partial purification and characterization of a repressor of ferritin mRNA translation. Proc Natl Acad Sci USA 85 9503-9507... 

Sonenberg, N., Rupprecht, K. M., Hecht, S. M., and Shatkin, A. J., 1979, Eukaryotic mRNA cap binding protein Purification by affinity chromatography on Se-pharose-coupled m GDP, Proc. Natl. Acad. Sci. USA 76 4345. 

In eukaryotes RNA is synthesized by three different RNA polymerases, each of which mediates the synthesis of a distinct product. RNA polymerase I (or A) synthesizes 25S and 18S rRNA. RNA polymerase II (or B) produces mRNA, whereas RNA polymerase III (or C produces t(ransfer)RNA and 5S rRNA. Each enzyme has its own characteristics that can be used to follow the purification of the individual enzymes. Sensitivity to a-amanitin is a useful property to characterize the polymerases. RNA polymerase II is highly sensitive to this toxin, whereas polymerase I is insensitive. Polymerase III varies in sensitivity from species to species but is in general less sensitive than polymerase II. 

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