Polyadenylation

The 3 -end of an eukaryotic mRNA is formed post-transcriptionally by endonucleolytic cleavage downstream of the coding sequence followed by extension of the upstream fragment by approximately 200 adenylate residues. In higher eukaryotes the specificity of the reaction depends on two conserved sequences in the pre-mRNA a highly conserved AAUAAA 10-30 nucleotides upstream from the cleavage site and a G/U-rich sequence within 50 nucleotides downstream from this site. The reaction requires a complex set of nuclear proteins, and a reconstituted polyadenylation reaction from pure proteins has not yet been achieved. However, processing of 

Formamide load buffer 80% (v/v) formamide 0.1% xylene cyanol 0.1% bromophenol blue 1 mM EDTA, pH 8.0 ATP, 25 mM 

RNA substrate (1-50 fmol 1000-10 000 cpm/fmol)a HeLa cell nuclear extract in buffer Db Proteinase K, 10 mg/ml (Merck) 

Mix by pipetting and incubate the reaction at 30°C for 30 min. Take out 5-10 p,l of the reaction for analysis of complex formation in native gels, if desired. 1 

Stop the reaction by adding 180 pi of proteinase K buffer supplemented with 5 p,g E. coli tRNA and 50 p,g Proteinase K. Incubate at 30°C for 10 min. 

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Ara-A is phosphorylated in mammalian cells to ara-AMP by adenosine kinase and deoxycytidine kinase. Further phosphorylation to the di- and triphosphates, ara-ADP and ara-ATP, also occurs. In HSV-1 infected cells, ara-A also is converted to ara-ATP. Levels of ara-ATP correlate directly with HSV rephcation. It has recently been suggested that ara-A also may exhibit an antiviral effect against adenovims by inhibiting polyadenylation of viral messenger RNA (mRNA), which may then inhibit the proper transport of the viral mRNA from the cell nucleus. 

Finally, this newly formed 3 terminal is polyadenylated in the nucleoplasm, as described below. 

Figure 37-13. Mechanisms of alternative processing of mRNA precursors. This form of RNA processing involves the selective inclusion or exclusion of exons, the use of alternative 5 donor or 3 acceptor sites, and the use of different polyadenylation sites.

In addition to affecting the efficiency of promoter utilization, eukaryotic cells employ alternative RNA processing to control gene expression. This can result when alternative promoters, intron-exon splice sites, or polyadenylation sites are used. Occasionally, heterogeneity within a cell results, but more commonly the same primary transcript is processed differendy in different tissues. A few examples of each of these types of regulation are presented below. 

Figure 39-19. Structure of a typical eukaryotic mRNA showing elements that are involved in regulating mRNA stability. The typical eukaryotic mRNA has a 5 noncoding sequence (5 NCS), a coding region, and a 3 NCS. All are capped at the 5 end, and most have a polyadenylate sequence at the 3 end. The 5 cap and 3 poly(A) tail protect the mRNA against exonuclease attack. Stem-loop structures in the 5 and 3 NCS, features in the coding sequence, and the AU-rich region in the 3 NCS are thought to play roles in mRNA stability.

Polynucleotides. Polynucleotides are potent interferon inducers. A mismatched, double-stranded synthetic polyribonucleotide ampligen and the double-stranded acids, polyadenylic-polyuridylic acid and polyinosinic-polycytidylic acids have been widely studied for cancer therapy(ii). Although these materials elicit excellent activity with murine rodents, therapeutic effects are dramatically decreased within primates. 

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.

Fig. 4. Schematic representation of expression of the rat pyruvate kinase (PK) gene. Exons specific to each isozyme are indicated by marked boxes. The exons common to Mj- and M2-type PK and common to L- and R-type PK are shown by open boxes. CAAT, CAT box TATA, TATA box AATAAA, polyadenylation signal.

M32. Mukai, T Yatsuki, H., Arai, Y., Joh, K., Matsuhashi, S and Hori, K., Human aldolase B gene Characterization of the genomic aldolase B gene and analysis of sequences required for multiple polyadenylations. J. Biochem. 102, 1043-1051 (1987). 

The polyadenylation of c-mos mRNA in maturing oocytes is similar to that of other maternal RNAs that are accumulated during oocyte... 

Figure 1. Expression of c-mos in mouse oocytes, c-mos is transcribed during oocyte growth and transcripts with short poly(A) tails are accumulated in fully-grown germinal vesicle (GV) stage oocytes. These transcripts are polyadenylated and translated following the resumption of meiosis and then degraded following fertilization and cleavage to the two-cell stage.

Vassalli, J. D., Huarte, J., Belin, D., Gubler, P., Vassalli, A., O Connell, M. L., Parton, L. A., Rickies, R. J., and Strickland, S. (1989). Regulated polyadenylation controls mRNA translation during meiotic maturation of mouse oocytes. Genes Dev. 3 2163-2171. 

Qian Y-W, Erikson E, Mailer JL 1998a Purification and cloning of a protein kinase that phosphorylates and activates the polo-like kinase Plxl. Science 282 1701-1704 Qian Y-W, Li C, Erikson E, Mailer JL 1998b Activated polo-like kinase Plxl is required at multiple points during mitosis in Xenopus laevis. Mol Cell Biol 18 4262-4271 Richter JD 1999 Cytoplasmic polyadenylation in development and beyond. Microbiol Mol Biol Rev 63 446-456... 

After more than 20 years, Walde et al. (1994) returned in a way to coacervate experiments, although using other methods. Walde (from the Luisi group) repeated nucleotide polymerisation of ADP to give polyadenylic acid, catalysed by polynucleotide phosphorylase (PNPase). But instead of Oparin s coacervates, the Zurich group used micelles and self-forming vesicles. They were able to demonstrate that enzyme-catalysed reactions can take place in these molecular structures, which can thus serve as protocell models. Two different supramolecular systems were used ... 

At this stage, mRNA can be polyadenylated using the poly(A) tailing kit (Ambion), according to the manufacturer s instructions. 

Example experiments using the previous methodologies are shown in Fig. 6.1. The major mRNA constructs described in this chapter are dia-grammatically represented in Fig. 6. IB and an example of in vitro transcribed and polyadenylated R-luc-4 sites mRNA is shown in Fig. 6.1A. In these experiments, translation of R-luc-4 sites mRNA is synergistically promoted by the physiological cap structure and the poly (A) tail (Fig. 6.1C), and full miR-dependent translational repression requires the presence of both modifications (Fig. 6.ID, Humphreys etal., 2005). (TheEMCV IRES-containing constructs are discussed later.)... 

Salles, F. J., and Strickland, S. (1995). Rapid and sensitive analysis of mRNA polyadenylation states by PCR. PCR Methods and Applications 4, 317-321. 

Huang, Y. S., Jung, M. Y., Sarkissian, M., and Richter, J. D. (2002). N-methyl-D-aspartate receptor signaling results in Aurora kinase-catalyzed CPEB phosphorylation and alpha CaMKII mRNA polyadenylation at synapses. EMBOJ. 21, 2139-2148. 

Wu, L., Wells, D., Tay, J., Mendis, D., Abbott, M. A., Barnitt, A., Quinlan, E., Heynen, A., Fallon, J. R., and Richter, J. D. (1998). CPEB-mediated cytoplasmic polyadenylation and the regulation of experience-dependent translation of alpha-CaMKII mRNA at synapses. Neuron 21, 1129-1139. 

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