MRNA polyadenylation

Fig. 1 Schematic outline of procedures employed in the synthesis of a cDNA gene copy from a polyadenylated mRNA template, insertion of the cDNA into a bacterial plasmid vector by a homopolymer tailing strategy, and cloning of the recombinant plasmid in an Escherichia coli host.

GAA-071, Endo and Sawasaki systematically modified the length and sequence of the 3 -UTR that works synergistically with the 5 -UTR to enhance transcript stability and initiation of translation. It was found that the activity of transcripts were more sensitive to the length of the 3 -UTR than to variations in the 3 -UTR sequence. mRNA synthesized with a 1626-nucleotide 3 -UTR, and the GAA-071 5 -UTR translation enhancer proved to be almost as active as the capped and polyadenylated mRNA transcribed from the unmodified pSP65 vector. 

Polyuridine has been coupled to agarose via an aminoethylcarbamoyl-dextran spacer to yield a new and highly effective biospecific adsorbent for affinity chromatography of polyadenylated mRNA. ... 

Poly(uridine) has been immobilized on agarose via a glycogen hydrazido-succinyl spacer arm and used for the affinity chromatographic purification of polyadenylated mRNA. ... 

The mRNA from the low CO -grown wild-type cells was then used to make a cDNA library. The cDNA was then cloned into the EcoRI site of the pUC-18 plasmid and these recombinant plasmids were used to transform E. coli strain JM83. Colonies carrying C. reinhardtii DNA that is expressed under low CO, conditions were screened for by differential hybridization to P-polyadenylated mRNA from both low C02-grown, wild-type cells and high C02-grown, CIA-5 cells (Figure 4). Transformants that preferentially hybridized to the wild type mRNA have been selected and will be further characterized. 

For cleaner results, polyadenylated RNA can be isolated as mentioned in the libraries chapter (Chapter 39). However, large amounts of RNA are required to obtain polyadenylated mRNA, so this choice is limited by the availability of the sample. Also, the time and effort employed in the extraction of more material do not necessarily correlate with an equal increase in performance. Therefore, total RNA will be the starting material used in these protocols. 

Poly(A) polymerases are found in a wide variety of organisms from higher animals and plants to yeast, bacteria, and certain viruses (1—3). In eukaryotes, the enzymes are presumed to play a key role in the polyadenylation of pre-mRNAs in the nucleus. However, in prokaryotes such as E. coli where polyadenylated mRNAs are minor species, the function of poly(A) polymerases remains a matter of speculation. In most species, poly(A) polymerases are single polypeptide ( 60 kDa) enzymes and catalyze a linear polymerization reaction as follows (Scheme 7.1). 

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