Many eukaryotic and viral mRNAs are modified at their 5' ends by addition of 7-Methylguanosine (N7-methyl guanosine or m7G), known as "Cap". "Capping" of the mRNA structure plays a crucial role in a variety of cellular processes which include translation initiation[1], splicing[2], intracellular transport[3] and turnover[4]. Capped mRNAs are generally more efficiently translated in wheat germ and reticulocyte in vitro translation systems[5], and they are less susceptible to exonuclease degradation during microinjection experiments compared to uncapped mRNAs[6].
Cap analogs are enzymatically incorporated at the RNA 5'-end by bacteriophage T7 RNA Polymerase-mediated in vitro Transcription.
| G-initiating promotor (T7 class III phi 6.5) | A-initiating promotor (T7 class II phi 2.5) |
|---|---|
| GP3G | GP3A |
| m7GP3G | m7GP3A |
| m27,3'-OGP3G (ARCA) |
[1] Gingras et al. (1999) eIF4 initiation factors: Effectors of mRNA recruitment to ribosomes and regulators of translation. Annu. Rev. Biochem. 68:913.
[2] Izaurralde et al. (1994) A nuclear cap binding protein complex involved in pre-mRNA splicing. Cell 78:657.
[3] Izaurralde et al. (1992) A cap binding protein that may mediate nuclear export of RNA polymerase II-transcribed RNAs. J. Cell Biol. 118:1287.
[4] Beelman et al. (1998) An essential component of the decapping enzyme required for normal rates of mRNA turnover. Nature 382:642.
[5] Paterson et al. (1979) Efficient translation of prokaryotic mRNAs in a eukaryotic cell-free system requires addition of a cap structure. Nature 279:692.
[6] Drummond et al. (1985) The effect of capping and polyadenylation on the stability, movement and translation of synthetic messenger RNAs in Xenopus oocytes. Nucl. Acids Res. 13:375.
[7] Pasquinelli et al. (1995) Reverse 5' caps in RNAs made in vitro by phage RNA polymerases. RNA 1:957.
[8] Grudzien et al. (2007) Synthesis of Anti-Reverse Cap Analogs (ARCAs) and their Applications in mRNA Translation and Stability. Methods Enzymol. 431:203.
